Multimodal trail molecules and uses in cellular therapies

ABSTRACT

Described herein are novel compositions comprising multimodal TRAIL agents and cells engineered to express such multimodal TRAIL agents, including cells encapsulated in a scaffold or matrix, for use in the treatment of disorders such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. Ser. No.15/225,202 filed on Aug. 1, 2016, which is a Divisional Application ofU.S. Ser. No. 13/982,343 filed on Oct. 24, 2013, which is 35 U.S.C. §371 National Phase Entry Application of International Application No.PCT/US2012/023221 filed Jan. 31, 2012, which designates the U.S., andwhich claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional PatentApplication Ser. No. 61/437,843 filed on Jan. 31, 2011, the contents ofwhich are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with Government support under R21 CA131980awarded by the National Institutes of Health (NIH). The Government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 1, 2016, isnamed 030258-069244-US_SL.txt and is 5,339 bytes in size.

FIELD OF THE INVENTION

The field of the invention relates to multimodal TRAIL molecules andcells engineered to express multimodal therapeutic agents, such asTRAIL, for use in cellular therapies.

BACKGROUND

Cancer remains one of the most deadly threats to human health. In theU.S., cancer affects nearly 1.3 million new patients each year, and isthe second leading cause of death after heart disease, accounting forapproximately 1 in 4 deaths. It is also predicted that cancer maysurpass cardiovascular diseases as the number one cause of death withinthe next decade. Solid tumors are responsible for most of those deaths.Although there have been significant advances in the medical treatmentof certain cancers, the overall 5-year survival rate for all cancers hasimproved only by about 10% in the past 20 years. Cancers, or malignanttumors, metastasize and grow rapidly in an uncontrolled manner, makingtimely detection and treatment extremely difficult.

Glioblastoma (GBM) is the most frequent and aggressive type of tumor todevelop from neuroepithelial tissue. GBMs are very heterogeneous withmultiple clones that contain varied genetic imbalances within onetumour, making it very difficult to treat successfully. Even withimproved surgical techniques and post-operative radiotherapy, the meanoverall survival time of patients with GBM after neurosurgical debulkingand radiotherapy is still limited to approximately 12 months. Currently,most chemotherapeutic agents have minimal effects on patient survival(J. M. A. Kuijlen et al., Neuropathology and Applied Neurobiology, 2010Vol. 36 (3), pp. 168-182).

SUMMARY OF THE INVENTION

The compositions and methods comprising multimodal TRAIL agentsdescribed herein provide novel therapeutic approaches for treatment ofcancers, such as glioblastoma. Shown herein is the development of novelmultifunctional, multimodal TRAIL agents as molecules that have bothdiagnostic (in vivo tracking via, for example, optical reporters) andtherapeutic (anti-tumor via a cytotoxic agent, e.g., TRAIL) properties.Further, their application in characterizing therapeutic delivery byengineered stem cells is demonstrated. These novel multimodal TRAILagents are easily optically visualized for serial monitoring ofcell-based pharmacokinetics, while retaining potent anti-tumorfunctions. Optical imaging permits elucidation of differences inpharmacokinetics, tissues distribution, and therapeutic efficacy of themultimodal TRAIL agents delivered to tumors by engineered stem cells orvia i.v. injection. Further, visualization of therapeutic levels of themultimodal TRAIL agents in real-time demonstrated for the first timethat a single administration of engineered neural stem cells expressinga multimodal TRAIL agent provides continuous sustained and localizeddelivery of the multimodal TRAIL agent that attenuates tumor growth,whereas a single i.v. infusion or direct administration of mediacontaining the multimodal TRAIL agent results in widespread off-targetbinding and significantly shortened delivery window that correlates withminimal anti-tumor effects.

Demonstrated herein are new approaches to treatment of tumors, such asGBM, using therapeutic stem cells encapsulated in biodegradable,synthetic extracellular matrix (sECM), using, for example, mouse modelsof human GBM resection. Using multimodal imaging, quantitative surgicaldebulking of human GBM tumors in mice that resulted in increasedsurvival is demonstrated. Next, as shown herein, sECM encapsulation ofengineered stem cells increased their retention in the tumor resectioncavity, permitted tumor-selective migration, and release of diagnosticand therapeutic proteins in vivo. Simulating the clinical scenario ofGBM treatment, the release of tumor-selective S-TRAIL (secretable tumornecrosis factor apoptosis inducing ligand) from sECM-encapsulated stemcells in the resection cavity eradicated residual tumor cells byinducing caspase-mediated apoptosis, delayed tumor regrowth, andsignificantly increased survival of mice. The studies described hereindemonstrate the efficacy and utility of encapsulated therapeutic stemcells in the treatment of cancers, such as GBM resection.

Accordingly, provided herein, in some aspects, are multimodal TRAILagents comprising a reporter module and a therapeutic TRAIL module,where the therapeutic TRAIL module comprises an extracellular domain ofhuman TRAIL.

In some embodiments of these aspects and all such aspects describedherein, the extracellular domain of human TRAIL comprises amino acids114-281 of SEQ ID NO: 1.

In some embodiments of these aspects and all such aspects describedherein, the multimodal TRAIL agent further comprises a signal sequence.In some such embodiments, the signal sequence comprises SEQ ID NO: 2.

In some embodiments of these aspects and all such aspects describedherein, the therapeutic TRAIL module further comprises an isoleucinezipper domain.

In some embodiments of these aspects and all such aspects describedherein, the multimodal TRAIL agent further comprises a linker domainC-terminal to the reporter module and N-terminal to the therapeuticTRAIL module. In some such embodiments, the linker domain comprises atleast eight amino acids. In some embodiments, the linker domaincomprises the amino acid sequence of SEQ ID NO: 4.

In another aspect, provided herein, are pharmaceutical compositionscomprising any of the multimodal TRAIL agents described hereincomprising a reporter module and a therapeutic TRAIL module, where thetherapeutic TRAIL module comprises an extracellular domain of humanTRAIL, and a pharmaceutically acceptable carrier.

In some aspects, provided herein are vectors comprising a nucleic acidsequence encoding any of the multimodal TRAIL agents comprising areporter module and a therapeutic TRAIL module described herein, wherethe therapeutic TRAIL module comprises an extracellular domain of humanTRAIL, or one or more modules thereof, described herein. In someembodiments of these aspects and all such aspects described herein, thevector is a lentiviral vector or an adenoviral vector.

Also provided herein, in some aspects, are cells comprising a nucleicacid sequence encoding any of the multimodal TRAIL agents comprising areporter module and a therapeutic TRAIL module described herein, wherethe therapeutic TRAIL module comprises an extracellular domain of humanTRAIL. In other aspects, provided herein are cells comprising any of thevectors comprising a nucleic acid sequence encoding any of themultimodal TRAIL agents, or one or more modules thereof, describedherein. In some embodiments of these aspects and all such aspectsdescribed herein, the cell is a stem cell. In some such embodiments, thestem cell is a neural stem cell or a mesenchymal stem cell. In someembodiments of these aspects and all such aspects described herein, thecell is encapsulated in a matrix or scaffold. In some embodiments, thematrix comprises a synthetic extracellular matrix. In some embodiments,the matrix is biodegradeable. In some embodiments, the syntheticextracellular matrix comprises a thiol-modified hyaluronic acid and athiol reactive cross-linker molecule. In some embodiments, the thiolreactive cross-linker molecule is polyethylene glycol diacrylate.

In some aspects, provided herein are compositions comprising an isolatedsomatic cell that comprises an exogenously introduced nucleic acidencoding any of the multimodal TRAIL agents comprising a reporter moduleand a therapeutic TRAIL module described herein operably linked to atleast one regulatory sequence.

In some embodiments of these aspects and all such aspects describedherein, the isolated somatic cell is an adult stem cell. In someembodiments, the adult stem cell is a neural stem cell or a mesenchymalstem cell. In some embodiments, the neural stem cell is generated from apluripotent stem cell.

In some embodiments of these aspects and all such aspects describedherein, the isolated somatic cell is encapsulated in a matrix orscaffold. In some embodiments, the matrix comprises a syntheticextracellular matrix. In some embodiments, the matrix is biodegradeable.In some embodiments, the synthetic extracellular matrix comprises athiol-modified hyaluronic acid and a thiol reactive cross-linkermolecule. In some embodiments, the thiol reactive cross-linker moleculeis polyethylene glycol diacrylate.

Also provided herein, in some aspects, are methods of treating a subjecthaving a malignant condition comprising administering a therapeuticallyeffective amount of any of the pharmaceutical compositions comprisingthe multimodal TRAIL agents described herein comprising a reportermodule and a therapeutic TRAIL module, where the therapeutic TRAILmodule comprises an extracellular domain of human TRAIL, and apharmaceutically acceptable carrier.

In other aspects, provided herein are methods of treating a subjecthaving a malignant condition comprising administering a therapeuticallyeffective amount of cells comprising a nucleic acid sequence encodingany of the multimodal TRAIL agents described herein comprising areporter module and a therapeutic TRAIL module, where the therapeuticTRAIL module comprises an extracellular domain of human TRAIL. In someembodiments of these methods and all such methods described herein, thecells are stem cells. In some such embodiments, the stem cell is aneural stem cell. In some embodiments of these methods and all suchmethods described herein, the cells are encapsulated in a matrix orscaffold. In some embodiments, the matrix comprises a syntheticextracellular matrix. In some embodiments, the matrix is biodegradeable.In some embodiments, the synthetic extracellular matrix comprises athiol-modified hyaluronic acid and a thiol reactive cross-linkermolecule. In some embodiments, the thiol reactive cross-linker moleculeis polyethylene glycol diacrylate.

In some aspects, provided herein are methods of treating a subjecthaving a malignant condition comprising administering a therapeuticallyeffective amount of cells comprising any of the vectors comprising anucleic acid sequence encoding any of the multimodal TRAIL agentscomprising a reporter module and a therapeutic TRAIL module, where thetherapeutic TRAIL module comprises an extracellular domain of humanTRAIL, or one or more modules thereof, described herein. In someembodiments of these methods and all such methods described herein, thecells are stem cells. In some such embodiments, the stem cell is aneural stem cell. In some embodiments of these methods and all suchmethods described herein, the cells are encapsulated in a matrix orscaffold. In some embodiments, the matrix comprises a syntheticextracellular matrix. In some embodiments, the matrix is biodegradeable.In some embodiments, the synthetic extracellular matrix comprises athiol-modified hyaluronic acid and a thiol reactive cross-linkermolecule. In some embodiments, the thiol reactive cross-linker moleculeis polyethylene glycol diacrylate.

In other aspects, provided herein are methods of treating a subjecthaving a malignant condition comprising administering a therapeuticallyeffective amount of a composition comprising an isolated somatic cellthat comprises an exogenously introduced nucleic acid encoding any ofthe multimodal TRAIL agents comprising a reporter module and atherapeutic TRAIL module described herein operably linked to at leastone regulatory sequence.

In some embodiments of these methods and all such methods describedherein, the isolated somatic cell is an adult stem cell. In someembodiments, the adult stem cell is a neural stem cell or a mesenchymalstem cell. In some embodiments, the neural stem cell is generated from apluripotent stem cell.

In some embodiments of these methods and all such methods describedherein, the isolated somatic cell is encapsulated in a matrix orscaffold. In some embodiments, the matrix comprises a syntheticextracellular matrix. In some embodiments, the matrix is biodegradeable.In some embodiments, the synthetic extracellular matrix comprises athiol-modified hyaluronic acid and a thiol reactive cross-linkermolecule. In some embodiments, the thiol reactive cross-linker moleculeis polyethylene glycol diacrylate.

In some embodiments of these methods and all such methods describedherein, the extracellular domain of human TRAIL comprises amino acids114-281 of SEQ ID NO: 1.

In some embodiments of these methods and all such methods describedherein, the multimodal TRAIL agent further comprises a signal sequence.In some such embodiments, the signal sequence comprises SEQ ID NO: 2.

In some embodiments of these methods and all such methods describedherein, the therapeutic TRAIL module further comprises an isoleucinezipper domain.

In some embodiments of these methods and all such methods describedherein, the multimodal TRAIL agent further comprises a linker domainC-terminal to the reporter module and N-terminal to the therapeuticTRAIL module. In some such embodiments, the linker domain comprises atleast eight amino acids. In some embodiments, the linker domaincomprises the amino acid sequence of SEQ ID NO: 4.

In some embodiments of these methods and all such methods describedherein, the malignant condition is a glioblastoma.

In some embodiments of these methods and all such methods describedherein, the methods further comprise administering to the subject one ormore additional chemotherapeutic agents, biologics, drugs, or treatmentsas part of a combinatorial therapy. In some such embodiments, thechemotherapeutic agent, biologic, drug, or treatment is selected fromthe group consisting of: radiation therapy, tumor resection surgery,gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479,vorinostat, rituximab, temozolomide, rapamycin, ABT-737, and PI-103.

In some embodiments of these methods and all such methods describedherein, the pharmaceutical compositions or cells are administered at asurgical site. In some embodiments, the surgical site is a tumorresection site.

Also provided herein, in some aspects, are pharmaceutical compositionscomprising any of the multimodal TRAIL agents described hereincomprising a reporter module and a therapeutic TRAIL module, where thetherapeutic TRAIL module comprises an extracellular domain of humanTRAIL, and a pharmaceutically acceptable carrier, for use in a method oftreating a malignant condition.

In some aspects, provided herein are vectors comprising a nucleic acidsequence encoding any of the multimodal TRAIL agents comprising areporter module and a therapeutic TRAIL module described herein, wherethe therapeutic TRAIL module comprises an extracellular domain of humanTRAIL, or one or more modules thereof, for use in a method of treating amalignant condition.

In some embodiments of these uses and all such uses described herein,the vector is a lentiviral vector or an adenoviral vector.

Also provided herein, in some aspects, are cells comprising a nucleicacid sequence encoding any of the multimodal TRAIL agents comprising areporter module and a therapeutic TRAIL module described herein, wherethe therapeutic TRAIL module comprises an extracellular domain of humanTRAI for use in a method of treating a malignant condition.

In other aspects, provided herein are cells comprising any of thevectors comprising a nucleic acid sequence encoding any of themultimodal TRAIL agents, or one or more modules thereof, describedherein for use in a method of treating a malignant condition. In someembodiments of these uses and all such uses described herein, the cellis a stem cell. In some such embodiments, the stem cell is a neural stemcell or a mesenchymal stem cell. In some embodiments of these methodsand all such methods described herein, the cell is encapsulated in amatrix or scaffold. In some embodiments, the matrix comprises asynthetic extracellular matrix. In some embodiments, the matrix isbiodegradeable. In some embodiments, the synthetic extracellular matrixcomprises a thiol-modified hyaluronic acid and a thiol reactivecross-linker molecule. In some embodiments, the thiol reactivecross-linker molecule is polyethylene glycol diacrylate.

In some aspects, provided herein are compositions comprising an isolatedsomatic cell that comprises an exogenously introduced nucleic acidencoding any of the multimodal TRAIL agents comprising a reporter moduleand a therapeutic TRAIL module described herein operably linked to atleast one regulatory sequence for use in a method of treating amalignant condition.

In some embodiments of these uses and all such uses described herein,the isolated somatic cell is an adult stem cell. In some embodiments,the adult stem cell is a neural stem cell or a mesenchymal stem cell. Insome embodiments, the neural stem cell is generated from a pluripotentstem cell.

In some embodiments of these uses and all such uses described herein,the isolated somatic cell is encapsulated in a matrix or scaffold. Insome embodiments, the matrix comprises a synthetic extracellular matrix.In some embodiments, the matrix is biodegradeable. In some embodiments,the synthetic extracellular matrix comprises a thiol-modified hyaluronicacid and a thiol reactive cross-linker molecule. In some embodiments,the thiol reactive cross-linker molecule is polyethylene glycoldiacrylate.

In some embodiments of these uses and all such uses described herein,the malignant condition is a glioblastoma.

Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

As used herein, the term “multimodal TRAIL agent” refers to a moleculecomprising at least two functional or biological activities—therapeuticand diagnostic. The therapeutic activity, e.g., cytotoxicity, isprovided by the therapeutic TRAIL module, as the term is defined herein.The diagnostic activity is provided by the reporter module, as the termis defined herein.

As used herein, the terms “therapeutic TRAIL module” or “therapeuticTRAIL variant” refer to a polypeptide, or a nucleotide sequence encodingsuch a polypeptide, comprising an extracellular domain of human TRAIL asdescribed in U.S. Pat. No. 6,284,236, the contents of which are hereinincorporated in their entirety by reference.

As used herein, the term “reporter module” referes to a molecule that isselected, designed, or engineered to permit in vivo monitoring andvisualization of the multimodal TRAIL agent. Preferably, the reportermodule permits minimally invasive monitoring and visualization of themultimodal TRAIL agent.

As used herein, the terms “secretion signal sequence,” “secretionsequence,” “secretion signal peptide,” or “signal sequence,” refer to asequence that is usually about 3-60 amino acids long and that directsthe transport of a propeptide to the endoplasmic reticulum and throughthe secretory pathway during protein translation.

As used herein, a “leucine zipper domain” refers to a naturallyoccurring or synthetic peptide that promotes oligomerization of theproteins in which it is found.

The terms “patient,” “subject,” and “individual” are usedinterchangeably herein, and refer to an animal, particularly a human, towhom it is desirable to administer a composition comprising a multimodalTRAIL agent or cells expressing a multimodal TRAIL agent. The term“subject” or “patient” as used herein also refers to human and non-humananimals. The term “non-human animals” includes all vertebrates, e.g.,mammals, such as non-human primates, (particularly higher primates),sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat,rabbits, cows, and any domestic animal or pet, as well as non-mammalssuch as chickens, amphibians, reptiles etc. In one embodiment, thesubject is human.

As used herein, the terms “patient sample” or “biological sample” refersto a fluid sample, a cell sample, a tissue sample or an organ sampleobtained from a patient. In some embodiments, a cell or population ofcells, or a quantity of tissue or fluid are obtained from a subject.Often, a “patient sample” will contain cells from the animal, but theterm can also refer to non-cellular biological material, such asnon-cellular fractions of blood, saliva, or urine. Biological samplesinclude, but are not limited to, tissue biopsies, scrapes (e.g. buccalscrapes), whole blood, plasma, serum, urine, saliva, cell culture,tissue biopsies, mucous membrane samples, feces, intestinal lavage,joint fluid, cerebrospinal fluid, a biliary sample, a respiratorysecretion, such as sputum, brochoalveolar lavage fluid sample, and thelike. A biological sample or tissue sample can refer to a sample oftissue or fluid isolated from an individual, including but not limitedto, for example, blood, plasma, serum, urine, stool, sputum, spinalfluid, pleural fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, andorgans. Samples can include frozen or paraffin-embedded tissue. The term“sample” includes any material derived by processing such a sample.Derived samples may, for example, include nucleic acids or proteinsextracted from the sample or obtained by subjecting the sample totechniques such as amplification or reverse transcription of mRNA,isolation and/or purification of certain components, etc.

The terms “stem cell” or “undifferentiated cell” as used herein, referto a cell in an undifferentiated or partially differentiated state thathas the property of self-renewal and has the developmental potential todifferentiate into multiple cell types, without a specific impliedmeaning regarding developmental potential (i.e., totipotent,pluripotent, multipotent, etc.). A stem cell is capable of proliferationand giving rise to more such stem cells while maintaining itsdevelopmental potential. In theory, self-renewal can occur by either oftwo major mechanisms. Stem cells can divide asymmetrically, which isknown as obligatory asymmetrical differentiation, with one daughter cellretaining the developmental potential of the parent stem cell and theother daughter cell expressing some distinct other specific function,phenotype and/or developmental potential from the parent cell. Thedaughter cells themselves can be induced to proliferate and produceprogeny that subsequently differentiate into one or more mature celltypes, while also retaining one or more cells with parentaldevelopmental potential. A differentiated cell may derive from amultipotent cell, which itself is derived from a multipotent cell, andso on. While each of these multipotent cells may be considered stemcells, the range of cell types each such stem cell can give rise to,i.e., their “developmental potential,” can vary considerably.Alternatively, some of the stem cells in a population can dividesymmetrically into two stem cells, known as stochastic differentiation,thus maintaining some stem cells in the population as a whole, whileother cells in the population give rise to differentiated progeny only.Accordingly, the term “stem cell” refers to any subset of cells thathave the developmental potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retain the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In some embodiments, the termstem cell refers generally to a naturally occurring parent cell whosedescendants (progeny cells) specialize, often in different directions,by differentiation, e.g., by acquiring completely individual characters,as occurs in progressive diversification of embryonic cells and tissues.Some differentiated cells also have the capacity to give rise to cellsof greater developmental potential. Such capacity may be natural or maybe induced artificially upon treatment with various factors. Cells thatbegin as stem cells might proceed toward a differentiated phenotype, butthen can be induced to “reverse” and re-express the stem cell phenotype,a term often referred to as “dedifferentiation” or “reprogramming” or“retrodifferentiation” by persons of ordinary skill in the art.

The term “somatic stem cell” is used herein to refer to any pluripotentor multipotent stem cell derived from non-embryonic tissue, includingfetal, juvenile, and adult tissue. Natural somatic stem cells have beenisolated from a wide variety of adult tissues including blood, bonemarrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle,and cardiac muscle. Exemplary naturally occurring somatic stem cellsinclude, but are not limited to, neural stem cells, neural crest stemcells, mesenchymal stem cells, hematopoietic stem cells, and pancreaticstem cells.

As used herein, the term “somatic cell” refers to any cell other than agerm cell, a cell present in or obtained from a pre-implantation embryo,or a cell resulting from proliferation of such a cell in vitro. Statedanother way, a somatic cell refers to any cell forming the body of anorganism, as opposed to a germline cell.

The term “expression” refers to the cellular processes involved inproducing RNA and proteins and as appropriate, secreting proteins,including where applicable, but not limited to, for example,transcription, translation, folding, modification and processing.“Expression products” include RNA transcribed from a gene, andpolypeptides obtained by translation of mRNA transcribed from a gene. Insome embodiments, an expression product is transcribed from a sequencethat does not encode a polypeptide, such as a microRNA.

The terms “isolated” or “partially purified” as used herein refers, inthe case of a nucleic acid or polypeptide, to a nucleic acid orpolypeptide separated from at least one other component (e.g., nucleicacid or polypeptide) that is present with the nucleic acid orpolypeptide as found in its natural source and/or that would be presentwith the nucleic acid or polypeptide when expressed by a cell, orsecreted in the case of secreted polypeptides. A chemically synthesizednucleic acid or polypeptide or one synthesized using in vitrotranscription/translation is considered “isolated.”

The term “transduction” as used herein refers to the use of viralparticles or viruses to introduce exogenous nucleic acids into a cell.

The term “transfection” as used herein refers the use of methods, suchas chemical methods, to introduce exogenous nucleic acids, such as thenucleic acid sequences encoding the multimodal TRAIL agents describedherein, into a cell. As used herein, the term transfection does notencompass viral-based methods of introducing exogenous nucleic acidsinto a cell. Methods of transfection include physical treatments(electroporation, nanoparticles, magnetofection), and chemical-basedtransfection methods. Chemical-based transfection methods include, butare not limited to, cyclodextrin, polymers, liposomes, nanoparticles,cationic lipids or mixtures thereof (e.g., DOPA, Lipofectamine andUptiFectin), and cationic polymers, such as DEAE-dextran orpolyethylenimine.

The term “anti-cancer therapy” refers to a therapy useful in treating amalignancy or cancer. Examples of anti-cancer therapeutic agentsinclude, but are not limited to, e.g., surgery, chemotherapeutic agents,growth inhibitory agents, cytotoxic agents, agents used in radiationtherapy, anti-angiogenesis agents, apoptotic agents, anti-tubulinagents, and other agents to treat cancer, such as anti-HER-2 antibodies(e.g., Herceptin®), anti-CD20 antibodies, an epidermal growth factorreceptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor),HER1/EGFR inhibitor (e.g., erlotinib (Tarceva®)), platelet derivedgrowth factor inhibitors (e.g., Gleevec™ (Imatinib Mesylate)), a COX-2inhibitor (e.g., celecoxib), interferons, cytokines, antagonists (e.g.,neutralizing antibodies) that bind to one or more of the followingtargets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGFreceptor(s), TRAIL/Apo2, and other bioactive and organic chemicalagents, etc. Combinations thereof are also included in the invention.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins, such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

As used herein, the terms “chemotherapy” or “chemotherapeutic agent”refer to any chemical agent with therapeutic usefulness in the treatmentof diseases characterized by abnormal cell growth. Such diseases includetumors, neoplasms and cancer as well as diseases characterized byhyperplastic growth. Chemotherapeutic agents as used herein encompassboth chemical and biological agents. These agents function to inhibit acellular activity upon which the cancer cell depends for continuedsurvival. Categories of chemotherapeutic agents includealkylating/alkaloid agents, antimetabolites, hormones or hormoneanalogs, and miscellaneous antineoplastic drugs. Most if not all ofthese agents are directly toxic to cancer cells and do not requireimmune stimulation. In one embodiment, a chemotherapeutic agent is anagent of use in treating neoplasms such as solid tumors. In oneembodiment, a chemotherapeutic agent is a radioactive molecule. One ofskill in the art can readily identify a chemotherapeutic agent of use(e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 inHarrison's Principles of Internal Medicine, 14th edition; Perry et al.,Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2.sup.nd ed.,.COPYRGT. 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds):Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-YearBook, 1995; Fischer D S, Knobf M F, Durivage H J (eds): The CancerChemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Themultimodal TRAIL agents described herein can be used in conjunction withadditional chemotherapeutic agents.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone time administration and typical dosages range from 10 to 200 units(Grays) per day.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with, a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorderassociated with a malignant condition or cancer. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.Alternatively, treatment is “effective” if the progression of a diseaseis reduced or halted. That is, “treatment” includes not just theimprovement of symptoms or markers, but also a cessation of at leastslowing of progress or worsening of symptoms that would be expected inabsence of treatment. Beneficial or desired clinical results include,but are not limited to, alleviation of one or more symptom(s) of amalignant disease, diminishment of extent of a malignant disease,stabilized (i.e., not worsening) state of a malignant disease, delay orslowing of progression of a malignant disease, amelioration orpalliation of the malignant disease state, and remission (whetherpartial or total), whether detectable or undetectable. The term“treatment” of a disease also includes providing relief from thesymptoms or side-effects of the disease (including palliativetreatment).

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, ““reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level or non-detectable level as compared to a referencesample), or any decrease between 10-100% as compared to a referencelevel.

The terms “increased”, “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the engineering and screening of multiple S-TRAIL andluciferase fusions in vitro. Schematic representations of lentiviraltransfer vectors bearing IRES-GFP cassettes and encoding various fusionsbetween secreted variant of the pro-apoptotic protein tumor necrosisfactor-related apoptosis-inducing ligand (S-TRAIL) and differentluciferase proteins are shown. Direct fusion variants: (1) TRAIL-Rluc,(2) TRAIL-Fluc, (3) TRAIL-GpLuc, (4) GpLuc-TRAIL. Variants to testintramolecular spacing: (5) GpLuc-linker 1-TRAIL, (6) GpLuc-linker2-TRAIL. Variants to test modification of secretion sequence: (7)SGpLuc-Linker 2-TRAIL, (8) SRlucO-linker 2-TRAIL. 293T cells weretransduced with lentiviral vectors encoding the designated fusionvariant. Bioluminescence imaging and enzyme-linked immunosorbent assaywere performed on conditioned medium from the transduced cells todetermine diagnostic luciferase activity or concentration of S-TRAIL,respectively. Therapeutic activity of each variant was determined byluciferase-based assay on human Gli36-EGFRvIII cells 24 hours afterincubation with equal volumes of conditioned media from lentiviraltransduced 293T cells. Abbreviations: GpL1TR, GpLuc-linker 1-TRAIL;GpL2TR, GpLuc-linker 2-TRAIL; GpTR, GpLuc-TRAIL; SGpL2TR, SGpLuc-Linker2-TRAIL; SRLOL2TR, SRlucO-linker 2-TRAIL; TRFL, TRAIL-Fluc; TRGp,TRAIL-GpLuc; TRRL, TRAIL-Rluc.

FIGS. 2A-2E depict screening S-TRAIL and luciferase fusion variants invivo. (2A): Western blot analysis of lysates from 293T cells transducedwith LV demonstrating expression of SGpL2TR and SRLOL2TR. (2B):Representative green fluorescent protein (GFP) photomicrograph (largemicrograph, 4×; inset, 10×) of human U251 glioma cells co-transducedwith equal MOI of lentiviral vectors (LV) encoding SGpL2TR, SRLOL2TR, orcontrol virus and GFP-FLuc. GFP appears as light spots. (2C-2D): Areaswith fluorescence appear as dark zones ringed with a lighter zone. U251glioma cells co-expressing GFP-FLuc and SGpL2TR, SRLOL2TR, or controlvirus were implanted subcutaneously in mice and imaged on days 1, 3, and15 to monitor secretion of TRAIL fusion proteins (GpLuc or RLucOintensities, (2C)) and on days 2, 7, and 15 to follow changes in tumorvolume (FLuc intensities, (2D)). (2E): Representative images and summarydata of similar experiments as those described in (2C and 2D) insteadusing nontherapeutic SGpLuc or SRLucO. Subcutaneous tumors were imagedon days 1, 5, and 10 for FLuc intensities to determine tumor volume orto monitor secretion of SGpLuc or SRLucO by coelenterazine injection.Representative day 10 images are shown. In all panels, *, p<0.05 versuscontrol. Abbreviations: SGpL2TR, SGpLuc-Linker 2-TRAIL; SRLOL2TR,SRlucO-linker 2-TRAIL; S-TRAIL, secreted variant of the pro-apoptoticprotein tumor necrosis factor-related apoptosis-inducing ligand.

FIGS. 3A-3J present imaging of SRLOL2TR that reveals differences in stemcell secretion and cancer cell killing. (3A): Representative images ofmNSC, hNSC, and mMSC transduced with LV encoding SRLOL2TR. GFP activityappears as lighter areas in panels other than top left which is abrightfield image. (3B): Summary data demonstrating differences intransduction efficiency between mNSC, hNSC, and mMSC 24 hourspost-transduction with increasing MOI of LV-SRLOL2TR. Green fluorescentprotein (GFP)-positive cells were counted and expressed as a ratio oftotal cell number for each stem cell type. (3C): Photon emission frommNSC, hNSC, and mMSC transduced with LV-SRLOL2TR were assayed at days 0,2, 7, and 14 post-transduction. (3D and 3E): Representative images andsummary graphs demonstrating the effects of different stem cell linessecreting SRLOL2TR co-cultured at increasing stem cell to tumor cellratios with Gli36-EGFRvIII (3D) or U251 human cancer cells. Greenfluorescence appears as the darkest grey, red fluorescence as the mediumgrey, and the lightest zones are those where red and overlap. (3E).After 24 hours of co-culture, levels of SRLOL2TR secretion by the stemcells were visualized by RLucO bioluminescence imaging and tumor cellkilling was visualized by Fluc bioluminescence imaging and quantifiedusing a luminometer. Luminesence appears as lighter zones. (3F): Westernblot analysis of cell lysates from mNSC or Gli36-EGFRvIII tumor cellsdemonstrating the expression of DR4 in each cell line. (3G):Immunocytochemical analysis of undifferentiated mNSC stained with anantibody against NSC marker Nestin (a), or following 10 days ofdifferentiation using antibodies against glial fibrillary acidic protein(GFAP) (b) or Olig-2 (c). Green fluorescence appears as the darkestgrey, red fluorescence as the medium grey, and the lightest zones arethose where red and overlap. (3H): Representative photomicrographsdemonstrating the migration of transduced mNSC towards gliomas overtime. GFP-expressing mNSC were implanted 1 mm lateral to establishedGli36-EGFRvIII-FD intracranial gliomas. On days 2 (a), 5 (b), and 10 (c)post-mNSC, implantation mice were sacrificed, brains were removed andsectioned, and both mNSC and glioma volumes were visualized usingfluorescence confocal microscopy. Panels a and b: 4× magnification;Panel c: 10× magnification. (3I and 3J): Human Gli36-EGFRvIII gliomacells were incubated with conditioned media from mNSC transduced withcontrol vector, SRLOL2TR, or purified S-TRAIL and caspase-3/7 activity(I), cleaved caspase-8 levels (3J), and cleaved PARP levels (3J) weredetermined by luciferase-based caspase 3/7 assay (3I) and Western blotanalysis (3J). In all panels, *, p<0.05 versus control. Abbreviations:hNSC, human neural stem cells; mMSC, primary mouse mesenchymal stemcells; mNSC, primary mouse neural stem cells; SRLucOL2TR, SRlucO-linker2-TRAIL.

FIGS. 4A-4I demonstrate that delivery by engineered stem cells improvesSRLOL2TR pharmacokinetics in vivo. (4A): Representative images andsummary graphs showing SRLOL2TR levels when delivered to tumors byengineered stem cells. Gli36-EGFRvIII-FD glioma cells were implantedsubcutaneously in mice, and 24 hours later FLuc imaging was performed todemonstrate the localization of the tumor. 24 hours post-imaging, mNSCsecreting SRLOL2TR were injected around one of the established tumors,and SRLOL2TR imaging was performed to visualize the secretion ofSRLOL2TR. (4B): Summary graph showing the effects on tumor volume ofcontrol mNSC or mNSC secreting SRLOL2TR 48 hours after implantationaround established Gli36-EGFRvIII-FD tumors assessed by FLuc imaging.(4C): Ex vivo analysis of biodistribution of mNSC-delivered fusionproteins assessed by RLucO imaging of organs removed 1-hour postinjection of coelenterazine. (4D-4I): In vivo bioluminescence imaging ofconditioned medium from LV-SRLOL2TR transduced cells injected into micebearing established Gli36-EGFRvIII-FD subcutaneous tumors by i.v.infusion (4D) or direct intratumoral administration (4G) and analyzed atdifferent time points after coelenterazine injection. Ex vivobioluminescence imaging of organs and tumor tissue from mice 1-hourpost-injection of media administered by i.v. infusion (4E) or directinjection (4H) followed by coelenterazine. Forty-eight hours after mediainjection, Fluc imaging was performed to determine changes in tumorvolumes (4F, 4I). In all panels, *, p<0.05 versus control.Abbreviations: SRLOL2TR, SRlucO-linker 2-TRAIL. Luminesence appears asgrey areas or black areas ringed by grey.

FIGS. 5A-5D demonstrate that stem cells efficiently deliver SRLOL2TR toeradicate intracranial glioblastoma. (5A): Representative FLucbioluminescent images and summary data of mice implanted intracraniallywith mNSC transduced with LV-GFP-FLuc, mixed with Gli36-EGFRvIII, andserially imaged for 15 days. (5B-5D): mNSC were transduced with controlvector or SRLOL2TR, and implanted with Gli36 EGFRvIII-FD intracraniallyin mice. On days 2, 6, 9, and 12 post-implantation, SRLOL2TR mice wereinjected with coelenterazine and RLucO imaging was performed tovisualize SRLOL2TR secretion (5B). Mice were injected with D-Luciferinand FLuc imaging was performed to visualize changes in glioma on days 1,3, 6, 9, 13, and 21 post-implantation. (5C): Representative images andsummary data are shown. C=Control; T=SRLOL2TR. (5D):Immunohistochemistry was performed on sections from brains containingGFP-FLuc-expressing mNSC 4 days post-implantation. Representative mergedimages are shown of brain sections containing mNSC (light grey) andstained with antibodies (darker grey) against nestin (a, e, i), glialfibrillary acidic protein (GFAP) (b, f, j), Tuj-1 (c, g, k), or Ki67 (d,h, 1). Nb=normal brain; T=tumor. In all panels, *, p<0.05 versuscontrol. GFP is visualized as a light grey and LUC as a darker grey.Abbreviations: SRLOL2TR, SRlucO-linker 2-TRAIL.

FIGS. 6A-6D depict a linear map of LV transfer vector, correlation ofcell number, and in vitro photon emission. (6A) Schematicrepresentations of self-inactivating lentiviral transfer plasmid basedon HIV-1 (CS-CW2). The S-TRAIL and luciferase fusions were cloneddownstream of the CMV promoter and upstream of and IRES element drivingGFP expression. (6B) Representative photomicrograph showing transductionof 293T by LV encoding S-TRAIL and luciferase fusion proteins. (6C-6D)In vitro bioluminescence imaging showing the correlation between cellnumber and FLuc expression (6C) or GpLuc/RLucO photon emission (6D) inU251 cells transduced with LV-GFP-FLuc and LV-SGpL2TR or LV-SRLOL2TR.

FIGS. 7A-7D depict the effects of altered signal sequence and in vivolight emission. (7A) Schematic representation of lentiviral transfervectors encoding fusions between Flt3L N-terminal signal sequence andGpLuc or SRLO. (7B-7D) Summary data showing the linear correlationbetween cell number and photon emission and representative fluorescentimages of cells transduced with FLuc-DsRed2 and either SGp or SRLO oftransduced cells.

FIGS. 8A-8B depict mNSC secretion and characterization ofGli36-EGFRvIII-FD fluorescence. (8A) Summary graph demonstratingdifferences in the levels of SRLOL2TR secretion from equally transducedmNSC, hNSC, and mMSC. After transduction with LV-SRLOL2TR, mNSC (MOI 2),hNSC (MOI 2), and mMSC (MOI 8) were plated at increasing cell numbers.Secretion levels were determined by visualization of SRLOL2TR levels inequal volumes of conditioned media by bioluminescence imaging. (8B)Representative images of Gli36-EGFRvIII expressing FLuc-DsRed2.

FIGS. 9A-9C depict the linear correlation of Gli36-EGFRvIII-FD andmNSC-SRLOL2TR photon emission. (9A-9C) In vitro bioluminescence imagingshowing correlation of luciferase activity with different numbers ofGli36-EGFRvIII-FD cells (9A), mNSC-SRLOL2TR (9B) or mNSC-GFP-FLuc (9C)plated at increasing number.

FIGS. 10A-10J demonstrate that tumor resection prolongs survival of micebearing GBM. (10A-10B) Human U87 GBM cells were transduced withLV-Fluc-mCherry and 48 hrs later cells imaged for mCherry expression(light grey) and Fluc activity (dark grey). Photomicrograph of U87 cellsexpressing Fluc-mCherry (10A) and plot revealing the correlation betweenU87-Fluc-mCherry cell number and Fluc activity (10B) are shown.(10C-10F) A cranial window was established in mice and U87-Fluc-mCherrycells (7.5×10⁴ or 1.5×10⁵) were implanted in the cranial window. Lightimages of the mouse skull with skin removed (10C), drilled rim aroundthe cranial window (10D). Dashed circle indicates the tumor growing areain the cranial window. (10E-10F) Mice with established U87-Fluc-mCherryGBMs in the cranial window were injected with a blood pool agent,Anigiosense-750 and imaged by intravital microscopy. Photomicrographspre-(10E) and post-(10F) tumor resection are shown. (10G-10H)Photomicrographs of low (10G) and high (10H) magnification H&E stainingof brain sections showing tumor resection cavity. (10I) Plot of therelative mean Fluc signal intensity and representative images pre- andpost-tumor resection of mice implanted with 7.5×10⁴ (resected on day 14post implantation) or 1.5×10⁵ (resected on day 21 post implantation) GBMcells are shown (p<0.05 versus pre-resection for each group). (10J)Kaplan-Meier survival curves of mice with and without resectedU87-Fluc-mCherry tumors. Scale bars, 100 μm (10A, 10E, 10F, 10H) and 400μm (10G). Original magnifications: ×2 (10C) and ×4 (10D).

FIGS. 11A-11I depict the characterization of engineered mNSC inbiocompatible sECMs in vitro and in mouse models of GBM: (11A-11B)Photomicrographs of mNSC expressing GFP-Fluc (mNSC-GFP-Fluc) grown inmonolayers (11A) and encapsulated in sECM (11B). (11C) mNSCco-expressing GFP-Fluc and a secretable luciferase, Ss-Rluc(o)) wereencapsulated in sECM and cell proliferation and protein secretion werefollowed by simultaneous Fluc and Rluc imaging of cells and culturemedium respectively. Plots and representative images are shown. (11D)mNSC-GFP-Fluc in suspension or encapsulated in sECM were implantedintracranially and mice were imaged serially for mNSC survival by Flucactivity. Plot showing the mNSC survival when implanted in sECM versussuspension in the brain over a period of 4 weeks. Representative imagesfrom day 14 mice are shown (p<0.05 versus non-encapsulated mNSC). (11E)mNSC co-expressing GFP-Fluc and Ss-Rluc(o) were encapsulated in sECM,implanted intracranially and cell viability (Fluc signal) and proteinsecretion (Rluc signal) were followed by simultaneous Fluc and Rlucimaging in vivo respectively. Plot showing the ratio of Rluc signalintensity relative to Fluc signal intensity. Representative images fromday 7 mice are shown. (11F-11I) Mice bearing U87-mCherry-Fluc GBMs inthe cranial windows were implanted with mNSC-GFP-Rluc encapsulated insECMs 1 mm away from an established tumor. GFP appears as light grey,LUC as a darker grey. Mice were imaged by intravital microscopy andphotomicrographs showing mNSC and tumor cells on day 1 (11F) and on day4 (11G, 11H, 11I) post mNSC implantation. Scale bars: 100 μm (11A, 11B,11H, 11I) and 200 μm (11F, 11G). Original magnifications: ×20 (11A, 11Binsets). Data are mean±s.e.m.

FIGS. 12A-12G depict that mNSC expressing therapeutic S-TRAILupregulates caspase-3/7 and induces GBM cell death in vitro: (12A-12E)mNSC expressing Ss-Rluc(o) or S-TRAIL were encapsulated in sECMs andplaced in the culture dish containing human GBM cells U87-Fluc-mCherry(darker grey). Photomicrographs showing sECM encapsulated mNSC at 8(12A, 12C) and at 24 (12B, 12D) hrs. Plot showing the tumor cellviability (p<0.05 versus controls) and caspase-3/7 activation (p<0.05versus mNSC-S-TRAIL) over 24 hours when co-cultured with either sECMencapsulated mNSC-Ss-Rluc(o) or mNSC-S-TRAIL (12E). (12F) Western blotanalysis on GBM cells collected at 8 hrs post sECM encapsulatedmNSC-S-TRAIL placement in the culture dish. (12G) Representative imagesand summary graphs demonstrating the effect of the release of Di-S-TRAILfrom mNSC encapsulated sECM co-cultured with U87-mCherry-Fluc atincreasing stem cell to tumor cell ratios. After 24 hrs of co-culture,levels of Di-S-TRAIL were visualized by Rluc bioluminescence imaging andtumor cell viability was visualized by Fluc bioluminescence imaging.Magnification 10× (12A-12D, 12G). Data are mean±s.e.m.

FIGS. 13A-13G demonstrate that sECM encapsulated mNSC-S-TRAILtransplanted into the tumor resection cavity increase survival of mice:(13A-13C) mNSC-GFP-Fluc in suspension or encapsulated in sECM wereimplanted intracranially in the resection cavity of the mouse model ofresection and, injected with Angiosense-750 i.v. and mice were imaged byintravital microscopy and by serial Fluc bioluminescence imaging.Photomicrographs showing the light image of the resection cavitycontaining sECM encapsulated mNSC (outlined area) (13A) and fluorescent(13B) and IHC image of sECM encapsulated mNSC-GFP-Fluc implanted(outlined area) in the resection cavity (13C). (13B) Fluorescencephotomicrograph showing mNSCs targeting residual GBM cells in a tumorresection cavity with leaky vasculature. (13C) Hematoxylin and eosinimage of sECMencapsulated mNSC-GFP-Fluc cells implanted (outlined area)in the resection cavity. (13D) Higher magnification fluorescencephotomicrograph showing mNSCs targeting residual GBM cells indicated byarrows in a tumor resection cavity with leaky vasculature. (13E) Plotand representative figures of the relative mean Fluc signal intensity ofmNSC-GFP-Fluc in suspension or encapsulated in sECMs placed in the GBMresection cavity (p<0.05 versus encapsulated mNSC). (13F-13G)mNSC-S-TRAIL or mNSC-GFP-Rluc encapsulated in sECM or mNSC-S-TRAIL insuspension were implanted intracranially in the resection cavity of themouse model of resection and mice were followed for changes in tumorvolume by serial Fluc bioluminescence imaging after aminoluciferin andluciferin injections, respectively, and for survival. TRAIL mediatedcaspase-3/7 activation and changes in tumor volumes (13G) as assessed bybioluminescence imaging Kaplan Meier survival curves (13F) are shown.Magnification 4× (13A, 13B) 10× (13C, 13D). (In panel 13F, tumorvolumes: p<0.05 versus controls; and caspase 3/7 activity: p<0.05 versusmNSC-S-TRAIL)

FIGS. 14A-14C depict tumor resection. U87-Fluc-mCherry tumor cells wereimplanted in mice with cranial window. Light images of the establishedintracranial U87-Fluc-mCherry tumor (14A) and the resected tumor (14B)in a cranial window. (14C) Resected U87-mCherry-Fluc tumor specimenimaged for Fluc bioluminescence. Dotted circle indicates the tumorimplantation site (in 14A) and tumor resection site (in 14B).

FIGS. 15A-15B depict mNSC-sECM in vitro characterization: (15A) mNSCswere either transduced with LV-GFP-Fluc or LV-Ss-Rluc(o) and differentcell numbers were encapsulated in sECM and imaged for cell viability(Fluc activity) and protein secretion (Rluc(o)) activity. Plot revealingthe correlation between cell number and Fluc or Rluc activity is shown.(15B) mNSCs were either transduced with LV-Ss-Rluc(o) or LV-S-TRAIL anddifferent cell numbers were encapsulated in sECM and incubated in growthmedium and 24 h later ELISA for S-TRAIL was performed on conditionedmedium. Representative photomicrographs of encapsulated mNSCs expressingS-TRAIL or Ss-Rluc(o) and plot revealing the correlation between theS-TRAIL concentration in the medium and the number of encapsulatedmNSCs.

FIG. 16 demonstrates that mNSC expressing therapeutic S-TRAIL placed inthe resection cavity reduces the tumors mean Fluc signal intensity overtime. Mice were implanted with U87mCherry-Fluc glioma cells in a cranialwindow, resected and implanted with sECM encapsulated mNSC-S-TRAIL ormNSC-GFP-Rluc. Mice were followed for changes in tumor volume by serialFluc bioluminescence imaging. Plot reveals the % Fluc signal intensitypost tumor resection over 49 days. Representative images are shown.*p<0.05 versus controls at day 28; determined by students t test; dataare mean±s.e.m.

FIGS. 17A-17L demonstrate that ECM-encapsulated therapeutic human MSCshave anti-tumor effects on primary invasive human GBMs in vitro and invivo. (17A, 17B) Primary invasive GBM8-mCherry-Fluc cells grown asneurospheres in a collagen matrix (17A) and brain section of micebearing GBM8-mCherry-Fluc tumors, showing the highly invasive nature ofGBM8 (17B). Arrow, site of implantation; arrowheads, path of invasion.(17C-17G) hMSCs expressing GFP or S-TRAIL were encapsulated in sECM andplaced in a culture dish containing human GBM8-Fluc-mCherry cells. hMSCswere followed for migration out of sECM, and GBM8 cells were followedfor their response to S-TRAIL secreted by hMSCs. Photomicrographs showsECM-encapsulated hMSCs on the day of plating (17C, 17E) and 48 h afterplating (17D, 17F). (17G) GBM8 cell viability at different time pointsafter culturing with varying numbers of either sECM-encapsulatedhMSC-GFP (control) or hMSC-S-TRAIL (TRAIL) cells. *P<0.05 versus TRAILat 8 h, 16 h and 24 h. (17H-17J) Encapsulated hMSC-S-TRAIL or hMSC-GFPcells in sECM were implanted intracranially in the tumor resectioncavity of mice bearing GBM8-mCherry-Fluc cells and mice were followedfor changes in tumor volume by serial Fluc bioluminescence imaging andcorrelative immunohistochemistry. Plot and representative images showthe relative mean Fluc signal intensity from mice bearingsECM-encapsulated hMSC-GFP or hMSC-S-TRAIL cells. *P<0.05 versus control(17H). (17I, 17J) Low-magnification (17I) and high-magnification (17J)photomicrographs of serial brain sections of mice showing hMSCs on day 5after hMSC implantation in the GBM8 resection cavity. (17K, 17L)Representative images showing cleaved caspase-3 staining (purple) onbrain sections from mice implanted with hMSC-S-TRAIL cells (17K) andcontrol cells (17L) 5 d after treatment. Scale bars: 100 μm (17A,17C-17F, 17I), 200 μm (17B) and 50 μm (17J-17L). Data are mean±s.e.m.

FIG. 18 demonstrates sensitivity/resistance of GBM cells to TRAILmediated apoptosis: Different established GBM (LN229, Gli79, A172, U251,U87, Gli36vIII, LN319) and primary GBM (BT74, GBM4, GBM6, GBM18, andGBM8-EF) lines were incubated with different concentrations of S-TRAIL,and GBM cell viability was determined 24 h postincubation. Plotrevealing the percentage cell viability is shown. *p<0.05 versus controldetermined by ANOVA; data are mean±s.e.m.

FIG. 19 depicts a western blot analysis showing un-cropped Western blotsof the data shown in FIG. 12F.

FIG. 20 demonstrates engineered human GBM8 cells for in vitro and invivo studies. Regression analysis indicating linear correlation betweenprimary human GBM8 cells expressing Fluc-mCherry cell number and Flucactivity. Representative photomicrograph of GBM8-mCherry-Fluc cells inculture is shown. Data was derived from the experiments performed intriplicate.

DETAILED DESCRIPTION

Provided herein are novel, multimodal, therapeutic agents comprising atherapeutic, secretable domain, such as domain of tumor necrosisfactor—related apoptosis-inducing ligand (TRAIL), and a reporter domainfor use as multifunctional therapeutic and diagnostic agents in thetreatment of cancers, such as glioblastoma. As described herein, themultimodal therapeutic agents, such as multimodal TRAIL agents, areuseful in combinatorial therapies, such as with chemotherapy,radiotherapy, and surgical interventions. Activation of cell surfacereceptors, such as death receptors (DRs), by the multimodal therapeuticagents and cells expressing such agents, such as engineered stem cells,provided herein, represent attractive therapeutic strategies to promoteapoptosis of tumor cells through the activation of extrinsic andintrinsic apoptotic pathways.

As described herein, diagnostic and therapeutic murine neural stem cells(NSCs) encapsulated in soluble extracellular matrix (sECM) were testedin a murine model of GBM resection. As demonstrated herein, sECMencapsulation of mNSC engineered to secrete a multimodal TRAIL agentsignificantly increased retention time in the GBM resection cavity,permitted robust tumor-selective migration and allowed secretion ofanti-tumor multimodal TRAIL proteins from the sECM encapsulated stemcells in vivo. Mimicking the clinical scenario of GBM resection andsubsequent treatment, TRAIL-secreting sECM encapsulated mNSCtransplanted in the resection cavity eradicated residual tumor cells,delayed tumor re-growth and significantly increased survival of mice.Furthermore, we demonstrate herein that TRAIL-secretingsECM-encapsulated stem cells transplanted in the resection cavitysignificantly delayed tumor regrowth in mice bearing both established(U87) and primary invasive (GBM8) GBMs and significantly increasedsurvival of mice bearing established GBMs.

In this study sECMs were employed that are based on a thiol-modifiedhyaluronic acid (HA) and a thiol reactive cross-linker (polyethyleneglycol diacrylate) which provides biocompatibility, physiologicalrelevance, and customizability (Xu et al. Prostaglandin Other LipidMediat 2009). Additionally, release profiles of sECM used in this studywere ideal to permit both migratory stem cells and secreted therapeuticproteins to exit the sECM. sECM encapsulation dramatically increased thesurvival of mNSC in resection cavities as compared to non-sECMencapsulated cells over a period of 4 weeks. We demonstrate herein thatsECM encapsulated engineered mNSC are effective by way of increasing theconcentration of therapeutic stem cells at the site of tumor resectionto extend the drug exposure time to tumor cells.

The ability of TRAIL to selectively target tumor cells while remainingharmless to most normal cells makes it an attractive candidate for anapoptotic therapy for highly malignant brain tumors. However, sustainedlevels of TRAIL are key to improving the efficiency and potency ofTRAIL-based pro-apoptotic cancer therapy. The results described hereinconfirm that TRAIL is a potent inhibitor of brain tumor growth, and thatencapsulated mNSC-S-TRAIL cytotoxic therapy is highly efficient ininducing apoptosis in residual GBM cells following GBM resection.

The studies described herein reveal the fate and therapeutic efficacy ofengineered and sECM encapsulated mNSC in a mouse model of GBM resection.Using the compositions and methods described herein, advances can bemade in the way stem cells can be engineered and used clinically incancer patients, such as brain tumor patients. In some embodiments ofthe methods described herein, neurosurgical removal of the main tumormass at the time of surgery can be combined with implantation ofpatient's own reprogrammed cells or mesenchymal stem cells,therapeutically engineered with anti-tumor agent(s) and encapsulated insECM, into the resection cavity of the tumor. These cells would resultin killing of both residual and invasive tumor cells with the ultimategoal of improving patient outcomes.

TRAIL and Apoptosis

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) isnormally expressed on both normal and tumour cells as a non-covalenthomotrimeric type-II transmembrane protein (memTRAIL). In addition, anaturally occurring soluble form of TRAIL (solTRAIL) can be generateddue to alternative mRNA splicing or proteolytic cleavage of theextracellular domain of memTRAIL and thereby still retainingtumour-selective pro-apoptotic activity. TRAIL utilizes an intricatereceptor system comprising four distinct membrane receptors, designatedTRAIL-R1, TRAIL-R2, TRAIL-R3 and TRAIL-R4. Of these receptors, onlyTRAIL-R1 and TRAIL-2 transmit an apoptotic signal. These two receptorsbelong to a subgroup of the TNF receptor family, the so-called deathreceptors (DRs), and contain the hallmark intracellular death domain(DD). This DD is critical for apoptotic signalling by death receptors(J. M. A. Kuijlen et al., Neuropathology and Applied Neurobiology, 2010Vol. 36 (3), pp. 168-182).

Apoptosis is integral to normal, physiologic processes that regulatecell number and results in the removal of unnecessary or damaged cells.Apoptosis is frequently dysregulated in human cancers, and recentadvancements in the understanding of the regulation of programmed celldeath pathways has led to the development of agents to reactivate oractivate apoptosis in malignant cells. This evolutionarily conservedpathway can be triggered in response to damage to key intracellularstructures or the presence or absence of extracellular signals thatprovide normal cells within a multicellular organism with contextualinformation.

TRAIL activates the “extrinsic pathway” of apoptosis by binding toTRAIL-R1 and/or TRAIL-R2, whereupon the adaptor protein Fas-associateddeath domain and initiator caspase-8 are recruited to the DD of thesereceptors. Assembly of this “death-inducing signaling complex” (DISC)leads to the sequential activation of initiator and effector caspases,and ultimately results in apoptotic cell death. The extrinsic apoptosispathway triggers apoptosis independently of p53 in response topro-apoptotic ligands, such as TRAIL. TRAIL-R1 can induce apoptosisafter binding non-cross-linked and cross-linked sTRAIL. TRAIL-R2 canonly be activated by cross-linked sTRAIL. Death receptor binding leadsto the recruitment of the adaptor FADD and initiator procaspase-8 and 10to rapidly form the DISC. Procaspase-8 and 10 are cleaved into itsactivated configuration caspase-8 and 10. Caspase-8 and 10 in turnactivate the effector caspase-3, 6 and 7, thus triggering apoptosis.

In certain cells, the execution of apoptosis by TRAIL further relies onan amplification loop via the “intrinsic mitochondrial pathway” ofapoptosis. The mitochondrial pathway of apoptosis is a stress-activatedpathway, e.g., upon radiation, and hinges on the depolarization of themitochondria, leading to release of a variety of pro-apoptotic factorsinto the cytosol. Ultimately, this also triggers effector caspaseactivation and apoptotic cell death. This mitochondrial release ofpro-apoptotic factors is tightly controlled by the Bcl-2 family of pro-and anti-apoptotic proteins. In the case of TRAIL receptor signaling,the Bcl-2 homology (BH3) only protein ‘Bid’ is cleaved into a truncatedform (tBid) by active caspase-8. Truncated Bid subsequently activatesthe mitochondrial pathway.

TRAIL-R3 is a glycosylphosphatidylinositol-linked receptor that lacks anintracellular domain, whereas TRAIL-R4 only has a truncated andnon-functional DD. The latter two receptors are thought, without wishingto be bound or limited by theory, to function as decoy receptors thatmodulate TRAIL sensitivity. Evidence suggests that TRAIL-R3 binds andsequesters TRAIL in lipid membrane microdomains. TRAIL-R4 appears toform heterotrimers with TRAIL-R2, whereby TRAIL-R2-mediated apoptoticsignalling is disrupted. TRAIL also interacts with the soluble proteinosteoprotegerin

Diffuse expression of TRAIL has been detected on liver cells, bileducts, convoluted tubules of the kidney, cardiomyocytes, lung epithelia,Leydig cells, normal odontogenic epithelium, megakaryocytic cells anderythroid cells. In contrast, none or weak expression of TRAIL wasobserved in colon, glomeruli, Henle's loop, germ and Sertoli cells ofthe testis, endothelia in several organs, smooth muscle cells in lung,spleen and in follicular cells in the thyroid gland. TRAIL proteinexpression was demonstrated in glial cells of the cerebellum in onestudy. Vascular brain endothelium appears to be negative for TRAIL-R1and weakly positive for TRAIL-R2. With regard to the decoy receptors,TRAIL-R4 and TRAIL-R3 have been detected on oligodendrocytes andneurones.

TRAIL-R1 and TRAIL-R2 are ubiquitously expressed on a variety of tumourtypes. Importantly for the compositions and methods comprisingmultimodal TRAIL agents described herein, TRAIL-R1 and TRAIL-R2 are alsoexpressed in the tumour tissue from astrocytoma grade II andglioblastoma patients. In a study on 62 primary GBM tumour specimens,TRAIL-R1 and TRAIL-R2 were expressed in 75% and 95% of the tumours,respectively. Of note, a statistically significant positive associationwas identified between agonistic TRAIL receptor expression and survival.Highly malignant tumours express a higher amount of TRAIL receptors incomparison with less malignant tumours or normal tissue. In generalTRAIL-R2 is more frequently expressed on tumour cells than TRAIL-R1.

Accordingly, the term “Tumour necrosis factor-related apoptosis-inducingligand” or “TRAIL” as used herein refers to the 281 amino acidpolypeptide having the amino acid sequence of:MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG (SEQ ID NO: 1), as described by, e.g.,NP 003801.1, together with any naturally occurring allelic, splicevariants, and processed forms thereof. Typically, TRAIL refers to humanTRAIL. The term TRAIL, in some embodiments of the aspects describedherein, is also used to refer to truncated forms or fragments of theTRAIL polypeptide, comprising, for example, specific TRAIL domains orresidues thereof. Reference to any such forms of TRAIL can be identifiedin the application, e.g., by “TRAIL (39-281).” The amino acid sequenceof the human TRAIL molecule as presented in SEQ ID NO: 1 comprises anN-terminal cytoplasmic domain (amino acids 1-18), a transmembrane region(amino acids 19-38), and an extracellular domain (amino acids 39-281).The extracellular domain comprises the TRAIL receptor-binding region.TRAIL also has a spacer region between the C-terminus of thetransmembrane domain and a portion of the extracellular domain Thisspacer region, located at the N-terminus of the extracellular domain,consists of amino acids 39 through 94 of SEQ ID NO: 1. Amino acids 138through 153 of SEQ ID NO: 1 correspond to a loop between the 13 sheetsof the folded (three dimensional) human TRAIL protein.

Multimodal TRAIL Agents

Described herein multimodal TRAIL agents comprising a therapeutic,secretable domain, such as domain of tumor necrosis factor-relatedapoptosis-inducing ligand (TRAIL), and a reporter domain for use asmultifunctional therapeutic and diagnostic agents in the treatment ofcancers, such as glioblastoma. These multimodal TRAIL agents are noveltherapeutic tools for utilizing the apoptotic effects on cancer cellsmediated by TRAIL, either by administering these agents directly or viaexpression of these agents in engineered cells, such as stem cells.

Preclinical studies have illustrated the promise of targeting TRAILactivity and using TRAIL as a therapeutic reagent in vivo with no orminimal toxicity. A variety of recombinant tumour necrosisfactor-related apoptosis-inducing ligand (TRAIL) molecules and agonisticantibodies directed at TRAIL death receptors TRAIL-R1 and/or TRAIL-R1have been developed. A recombinant trimeric form of TRAIL is beingexplored in an ongoing multicentre clinical trail for B-CLL patients.Importantly, no significant side effects have been reported so far, thuscorroborating the safety of TRAIL treatment in humans. In addition, anumber of agonistic antibodies (e.g., HGS-ETR1, HGS-ETR2, HGS-TR2J,LBY135, CS-1008, AMG 655) that selectively target TRAIL-R1 or TRAIL-R2have been developed. All of these antibodies have potent tumouricidalactivity in vitro and in vivo and appear to have a low toxicity profilein early-phase clinical studies.

However, the efficacy of using naturally occurring soluble TRAIL ishampered by various factors, including rapid clearance from thecirculation by the kidney. Reports have demonstrated that soluble TRAILhas an approximate half-life of only 30 minutes in primates, and asimilar pharmacokinetic profile in humans in a phase I clinical trial(Ashkenazi A. et al., J Clin Oncol 2008; 26: 3621-30, and Kelley S K etal., J Pharmacol Exp Ther 2001; 299: 31-8, the contents of which areherein incorporated by reference in their entireties). Some reports haveattempted to overcome the rapid clearance by fusing soluble TRAIL withan antibody derivative, such as scFv245 (Bremer E. et al., J Mol Med2008; 86: 909-24; Bremer E, et al., Cancer Res 2005; 65: 3380-88; BremerE, et al., J Biol Chem 2005; 280: 10025-33, and Stieglmaier J, et al.,Cancer Immunol Immunother 2008; 57: 233-46).

One of the primary challenges to achieving effective anti-tumortherapies is highly efficient delivery of the anti-tumor agentspecifically to the tumor, while minimizing toxicity to nonmalignanttissue. Although simple to administer, systemic administration oftherapies can lead to accumulation of the toxic compounds at high levelsin the liver and kidneys, resulting in dose-limiting renal- andhepatotoxicity (Kelley et al. J Pharmacol Exp Ther 2001, Lin, Drug MetabDispos 1998). TRAIL has been shown to have minimal cytotoxic effects onnormal tissue; however, its short half-life and accumulation aftersystemic injection have been limitations to its potential use in clinics(Ashkenazi et al., J Clin Oncol 2008). Because of their potential tomigrate to sites of disease and integrate into the cytoarchitecture ofthe brain, stem cells (e.g., neural stem cells, mesenchymal stem cells)have received much interest for the treatment of numerous neurologicdisorders (Corsten and Shah, Lancet Oncology 2008, Singec et al. AnnuRev Med 2007). Previous studies from our lab and others demonstratedthat neural stem cells (NSCs) and human mesenchymal stem cells (MNCs)migrate extensively throughout the murine brain and exhibit an inherentcapacity to home to established gliomas (Sasportas et al. Proc Natl AcadSci 2009, Shah et al Ann Neurol 2005, Shah et al. J Neurosci 2008, thecontents of each of which are herein incorporated by reference in theirentireties). Stem cells armed with S-TRAIL inhibited progression ofgliomas in a xenogenic transplant model (Sasportas et al. Proc Natl AcadSci 2009, Shah et al Ann Neurol 2005); however, assessing thepharmacokinetics of the molecules released by therapeutic NSC has beendifficult.

Therapeutic TRAIL Modules

As demonstrated herein, the inventors have engineered multimodal TRAILagents that have both increased apoptosis-inducing abilities and can besecreted from a cell, such as a neural stem cell, and can be imaged inreal-time for diagnostic purposes, for use in the compositions andmethods described herein (K. Shah et al., Cancer Research 2004, 64:3236-3242; K. Shah et al., Molecular Therapy 2005, 11(6): 926-931; ShahK, et al., Ann Neurol 2005; 57: 34-41; Sasportas L S et al., Proc NatlAcad Sci USA 2009; 106: 4822-7; Shah K. et al., 1. J Neurosci 2008; 28:4406-13; Hingtgen S et al., Mol Cancer Ther 2008; 7: 3575-85; thecontents of each of which is herein incorporated by reference in theirentireties). These multimodal TRAIL agents comprise a therapeutic TRAILmodule or therapeutic TRAIL domain or variant thereof having TRAILapoptotic activity. As used herein, a “therapeutic TRAIL module,”“therapeutic TRAIL domain,” or “therapeutic TRAIL variant” refers to apolypeptide, or a nucleotide sequence encoding such a polypeptide,comprising an extracellular domain of human TRAIL, such as a human TRAILof SEQ ID NO: 1, and maintaining TRAIL apoptotic activity. In someembodiments of the aspects described herein, an N-terminal secretionsignal sequence is fused to the N-terminal of the extracellular domainof human TRAIL. In some embodiments of the aspects described herein, thetherapeutic TRAIL module can further comprise an isoleucine zipperdomain.

Accordingly, provided herein, in some aspects, are multimodal TRAILagents comprising a therapeutic human TRAIL domain or variant thereof,wherein the therapeutic human TRAIL domain comprises an extracellulrdomain of human TRAIL. In some embodiments of the aspects describedherein, the extracellular domain of human TRAIL comprises amino acids39-281 of SEQ ID NO: 1. In some embodiments of the aspects describedherein, the extracellular domain of human TRAIL comprises amino acids95-281 of SEQ ID NO: 1. In some embodiments of the aspects describedherein, the extracellular domain of human TRAIL comprises amino acids114-281 of SEQ ID NO: 1. In some embodiments of the aspects describedherein, the extracellular domain of human TRAIL comprises a sequencehaving at least 90% identity to amino acids 114-281 of SEQ ID NO: 1 andretains TRAIL apoptotic activity. In some embodiments of the aspectsdescribed herein, the extracellular domain of human TRAIL used in amultimodal TRAIL agent consists essentially of amino acids 114-281 ofSEQ ID NO: 1. In some embodiments of the aspects described herein, theextracellular domain of human TRAIL used in a multimodal TRAIL agentconsists of amino acids 114-281 of SEQ ID NO: 1.

Variants and derivatives of native TRAIL proteins for use in thetherapeutic TRAIL modules that retain a desired biological activity ofTRAIL, such as “TRAIL apoptotic activity” are also within the scope ofthe compositions and methods described herein. In some embodiments, thebiological or apoptotic activity of a therapeutic TRAIL module isessentially equivalent to the biological activity of a native TRAILprotein. In some such embodiments, biological activity of a native TRAILprotein is TRAIL apoptotic activity. One measurement of TRAIL apoptoticactivity by a TRAIL variant or TRAIL domain is the ability to induceapoptotic death of Jurkat cells. Assay procedures for identifyingbiological activity of TRAIL variants by detecting apoptosis of targetcells, such as Jurkat cells, are well known in the art. DNA laddering isamong the characteristics of cell death via apoptosis, and is recognizedas one of the observable phenomena that distinguish apoptotic cell deathfrom necrotic cell death. Apoptotic cells can also be identified usingmarkers specific for apoptotic cells, such as Annexin V, in combinationwith flow cytometric techniques, as known to one of skill in the art.Further examples of assay techniques suitable for detecting death orapoptosis of target cells include those described in the Examplessection.

TRAIL variants can be obtained by mutations of native TRAIL nucleotidesequences, for example. A “TRAIL variant,” as referred to herein, is apolypeptide substantially homologous to a native TRAIL, but which has anamino acid sequence different from that of native TRAIL because of oneor a plurality of deletions, insertions or substitutions. “TRAILencoding DNA sequences” encompass sequences that comprise one or moreadditions, deletions, or substitutions of nucleotides when compared to anative TRAIL DNA sequence, but that encode a TRAIL protein or fragmentthereof that is essentially biologically equivalent to a native TRAILprotein, i.e., has the same apoptosis inducing activity.

The variant amino acid or DNA sequence preferably is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or more, identical to a nativeTRAIL sequence. The degree of homology or percent identity) between anative and a mutant sequence can be determined, for example, bycomparing the two sequences using freely available computer programscommonly employed for this purpose on the world wide web.

Alterations of the native amino acid sequence can be accomplished by anyof a number of known techniques known to one of skill in the art.Mutations can be introduced, for example, at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes ananalog having the desired amino acid insertion, substitution, ordeletion. Alternatively, oligonucleotide-directed site-specificmutagenesis procedures can be employed to provide an altered nucleotidesequence having particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsinclude those disclosed by Walder et al. (Gene 42:133, 1986); Bauer etal. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19);Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press,1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are hereinincorporated by reference in their entireties.

TRAIL variants can, in some embodiments, comprise conservativelysubstituted sequences, meaning that one or more amino acid residues of anative TRAIL polypeptide are replaced by different residues, and thatthe conservatively substituted TRAIL polypeptide retains a desiredbiological activity, i.e., apoptosis inducing activity or TRAILapoptotic activity, that is essentially equivalent to that of the nativeTRAIL polypeptide. Examples of conservative substitutions includesubstitution of amino acids that do not alter the secondary and/ortertiary structure of TRAIL.

In other embodiments, TRAIL variants can comprise substitution of aminoacids that have not been evolutionarily conserved. Conserved amino acidslocated in the C-terminal portion of proteins in the TNF family, andbelieved to be important for biological activity, have been identified.These conserved sequences are discussed in Smith et al. (Cell, 73:1349,1993, see page 1353 and FIG. 6); Suda et al. (Cell, 75:1169, 1993, seeFIG. 7); Smith et al. (Cell, 76:959, 1994, see FIG. 3); and Goodwin etal. (Eur. J. Immunol., 23:2631, 1993, see FIG. 7 and pages 2638-39)hereby incorporated in their entireties by reference. Advantageously, insome embodiments, these conserved amino acids are not altered whengenerating conservatively substituted sequences. In some embodiments, ifaltered, amino acids found at equivalent positions in other members ofthe TNF family are substituted. Among the amino acids in the human TRAILprotein of SEQ ID NO:1 that are conserved are those at positions 124-125(AH), 136 (L), 154 (W), 169 (L), 174 (L), 180 (G), 182 (Y), 187 (Q), 190(F), 193 (Q), and 275-276 (FG) of SEQ ID NO:1. Another structuralfeature of TRAIL is a spacer region (i.e., TRAIL (39-94)) between theC-terminus of the transmembrane region and the portion of theextracellular domain that is believed to be important for biologicalapoptotic activity. In some embodiments, when the desired biologicalactivity of TRAIL domain is the ability to bind to a receptor on targetcells and induce apoptosis of the target cells substitution of aminoacids occurs outside of the receptor-binding domain.

A given amino acid of a TRAIL domain can, in some embodiments, bereplaced by a residue having similar physiochemical characteristics,e.g., substituting one aliphatic residue for another (such as Ile, Val,Leu, or Ala for one another), or substitution of one polar residue foranother (such as between Lys and Arg; Glu and Asp; or Gln and Asn).Other such conservative substitutions, e.g., substitutions of entireregions having similar hydrophobicity characteristics, are well known.TRAIL polypeptides comprising conservative amino acid substitutions canbe tested in any one of the assays described herein to confirm that adesired TRAIL apoptotic activity of a native TRAIL molecule is retained.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H).

Alternatively, naturally occurring residues can be divided into groupsbased on common side-chain properties: (1) hydrophobic: Norleucine, Met,Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;(3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues thatinfluence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Particularly preferred conservative substitutions for use in the TRAILvariants described herein are as follows: Ala into Gly or into Ser; Arginto Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln intoAsn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln;Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, intoGln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, intoLeu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp;and/or Phe into Val, into Ile or into Leu.

Any cysteine residue not involved in maintaining the proper conformationof the multimodal TRAIL agent also can be substituted, generally withserine, to improve the oxidative stability of the molecule and preventaberrant crosslinking. Conversely, cysteine bond(s) can be added to themultimodal TRAIL agent to improve its stability or facilitateoligomerization.

Signal Sequences

The multimodal TRAIL agents described herein can further comprise, insome embodiments, a secretion signal sequence that permits a cellengineered to express a multimodal TRAIL agent to secrete the agent. Asused herein, the terms “secretion signal sequence,” “secretionsequence,” “secretion signal peptide,” or “signal sequence,” refer to asequence that is usually about 3-60 amino acids long and that directsthe transport of a propeptide to the endoplasmic reticulum and throughthe secretory pathway during protein translation. As used herein, asignal sequence, which can also be known as a signal peptide, a leadersequence, a prepro sequence or a pre sequence, does not refer to asequence that targets a protein to the nucleus or other organelles, suchas mitochondria, chloroplasts and apicoplasts. In some embodiments ofthe multimodal TRAIL agents described herein, a “secretion signalsequence” comprises 5 to 15 amino acids with hydrophobic side chainsthat are recognized by a cytosolic protein, SRP (Signal RecognitionParticle), which stops translation and aids in the transport of anmRNA-ribosome complex to a translocon in the membrane of the endoplasmicreticulum. In some embodiments of the multimodal TRAIL agents describedherein, the signal peptide comprises at least three regions: anamino-terminal polar region (N region), where frequently positivecharged amino acid residues are observed, a central hydrophobic region(H region) of 7-8 amino acid residues and a carboxy-terminal region (Cregion) that includes the cleavage site. Commonly, the signal peptide iscleaved from the mature protein with cleavage occurring at this cleavagesite.

The secretory signal sequence is operably linked to the sequenceencoding the therapeutic TRAIL module of the multimodal TRAIL agentsdescribed herein, such that the two sequences are joined in the correctreading frame and positioned to direct the newly synthesized polypeptideinto the secretory pathway of the host cell. Secretory signal sequencesare commonly positioned 5′ to the nucleotide sequence encoding thepolypeptide of interest, although certain secretory signal sequences canbe positioned elsewhere in the nucleotide sequence of interest (see,e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat.No. 5,143,830).

In some embodiments of the aspects described herein, the secretorysequence comprises amino acids 1-81 of the following Flt3L amino acidsequence: MTVLAPAWSP NSSLLLLLLL LSPCLRGTPD CYFSHSPISS NFKVKFRELTDHLLKDYPVT VAVNLQDEKH CKALWSLFLA QRWIEQLKTV AGSKMQTLLE DVNTEIHFVTSCTFQPLPEC LRFVQTNISH LLKDTCTQLL ALKPCIGKAC QNFSRCLEVQ CQPDSSTLLPPRSPIALEAT ELPEPRPRQL LLLLLLLLPL TLVLLAAAWG LRWQRARRRG ELHPGVPLPS HP(SEQ ID NO: 2, GenBank Accession P49772), or a fragment thereof. In someembodiments of the aspects described herein, the signal peptidecomprises amino acids 1-81 of SEQ ID NO: 2. In some embodiments of theaspects described herein, the secretory signal sequence comprises asequence having at least 90% identity to amino acids 1-81 of SEQ ID NO:2. In some embodiments of the aspects described herein, the secretorysignal sequence consists essentially of amino acids 1-81 of SEQ ID NO:2. In some embodiments of the aspects described herein, the secretorysignal sequence consists of amino acids 1-81 of SEQ ID NO: 2.

While the secretory signal sequence can be derived from Flt3L, in otherembodiments a suitable signal sequence can also be derived from anothersecreted protein or synthesized de novo. Other secretory signalsequences which can be substituted for the Flt3L signal sequence forexpression in eukaryotic cells include, for example, naturally-occurringor modified versions of the human IL-17RC signal sequence, otPA pre-prosignal sequence, human growth hormone signal sequence, human CD33 signalsequence Ecdysteroid Glucosyltransferase (EGT) signal sequence, honeybee Melittin (Invitrogen Corporation; Carlsbad, Calif.), baculovirusgp67 (PharMingen: San Diego, Calif.) (US Pub. No. 20110014656).Additional secretory sequences include secreted alkaline phosphatasesignal sequence, interleukin-1 signal sequence, CD-14 signal sequenceand variants thereof (US Pub. No. 20100305002) as well as the followingpeptides and derivatives thereof: Sandfly Yellow related protein signalpeptide, silkworm friboin LC signal peptide, snake PLA2, Cyrpidinanoctiluca luciferase signal peptide, and pinemoth fibroin LC signalpeptide (US Pub. No. 20100240097). Further signal sequences can beselected from databases of protein domains, such as SPdb, a signalpeptide database described in Choo et al., BMC Bioinformatics 2005,6:249, LOCATE, a mammalian protein localization database described inSprenger et al. Nuc Acids Res, 2008, 36:D230D233, or identified usingcomputer modeling by those skilled in the art (Ladunga, Curr OpinBiotech 2000, 1:13-18).

Selection of appropriate signal sequences and optimization orengineering of signal sequences is known to those skilled in the art(Stern et al., Trends in Cell & Molecular Biology 2007 2:1-17; Barash etal., Biochem Biophys Res Comm 2002, 294:835-842). In some embodiments,signal sequences can be used that comprise a protease cleavage site fora site-specific protease (e.g., Factor IX or Enterokinase). Thiscleavage site can be included between the pro sequence and the bioactivesecreted peptide sequence, e.g., TRAIL domain, and the pro-peptide canbe activated by the treatment of cells with the site-specific protease(US Pub. No. 20100305002).

Leucine Zippers

The therapeutic TRAIL modules and multimodal TRAIL agents describedherein can, in some embodiments, further comprise a leucine zipperdomain sequence. As used herein, “leucine zipper domains” refer tonaturally occurring or synthetic peptides that promote oligomerizationof the proteins in which they are found. The leucine zipper is asuper-secondary structure that functions as a dimerization domain, andits presence generates adhesion forces in parallel alpha helices. Asingle leucine zipper comprises multiple leucine residues atapproximately 7-residue intervals, which forms an amphipathic alphahelix with a hydrophobic region running along one side. The dimer formedby a zipper domain is stabilized by the heptan repeat, designated(abcdefg). according to the notation of McLachlan and Stewart (J. Mol.Biol. 98:293; 1975), in which residues a and d are generally hydrophobicresidues, with d being a leucine, which line up on the same face of ahelix. Oppositely-charged residues commonly occur at positions g and e.Thus, in a parallel coiled coil formed from two helical zipper domains,the “knobs” formed by the hydrophobic side chains of the first helix arepacked into the “holes” formed between the side chains of the secondhelix. The residues at position d (often leucine) contribute largehydrophobic stabilization energies, and are important for oligomerformation (Krystek et al., Int. J. Peptide Res. 38:229, 1991). Thishydrophobic region provides an area for dimerization, allowing themotifs to “zip” together. Furthermore, the hydrophobic leucine region isabsolutely required for DNA binding. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, 1988), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize.

Examples of zipper domains are those found in the yeast transcriptionfactor GCN4 and a heat-stable DNA-binding protein found in rat liver(C/EBP; Landschulz et al., Science 243:1681, 1989). The nucleartransforming proteins, fos and jun, also exhibit zipper domains, as doesthe gene product of the murine proto-oncogene, c-myc (Landschulz et al.,Science 240:1759, 1988). The fusogenic proteins of several differentviruses, including paramyxovirus, coronavirus, measles virus and manyretroviruses, also possess zipper domains (Buckland and Wild, Nature338:547, 1989; Britton, Nature 353:394, 1991; Delwart and Mosialos, AIDSResearch and Human Retrovirtises 6:703, 1990). The zipper domains inthese fusogenic viral proteins are near the transmembrane region of theprotein. Oligomerization of fusogenic viral proteins is involved infusion pore formation (Spruce et al, Proc. Natl. Acad. Sci. U.S.A.88:3523, 1991). Zipper domains have also been reported to play a role inoligomerization of heat-shock transcription factors (Rabindran et al.,Science 259:230, 1993).

Examples of leucine zipper domains suitable for producing multimodalTRAIL agents include, but are not limited to, those described in PCTapplication WO 94/10308; U.S. Pat. No. 5,716,805; the leucine zipperderived from lung surfactant protein D (SPD) described in Hoppe et al.,1994, FEBS Letters 344:191; and Fanslow et al., 1994, Semin. Immunol.6:267-278, the contents of each of which are hereby incorporated byreference in their entireties. In some embodiments of the multimodalTRAIL agents, leucine residues in a leucine zipper domain are replacedby isoleucine residues. Such peptides comprising isoleucine can also bereferred to as isoleucine zippers, but are encompassed by the term“leucine zippers” as used herein.

Reporter Modules

The multimodal TRAIL agents described herein are engineered to have atleast two functional activities-therapeutic and diagnostic. Thetherapeutic activity is provided by the therapeutic TRAIL module, whichprovides the agent the ability to induce apoptosis of target cells andcytotoxicity. The diagnostic activity is provided by the reportermodule, which is selected, designed or engineered to permit in vivomonitoring and visualization of the multimodal TRAIL agent. Preferably,the reporter module permits minimally invasive monitoring andvisualization of the multimodal TRAIL agent, as described herein.

A “reporter module,” as used herein, refers to a molecule which providesan analytically identifiable signal allowing detection of a multimodalTRAIL agent by non-invasive means. Detection can be either qualitativeor quantitative. Commonly used reporter modules include, for example,fluorophores, enzymes, biotin, chemiluminescent molecules,bioluminescent molecules, digoxigenin, avidin, streptavidin, orradioisotopes. Commonly used enzymes include, for example, horseradishperoxidase, alkaline phosphatase, glucose oxidase andbeta-galactosidase, among others. Enzymes can be conjugated to avidin orstreptavidin for use in a reporter module. Similarly, reporter modulescan be conjugated to avidin or streptavidin for use with a biotinylatedenzyme. The substrates to be used with these enzymes are generallychosen for the production, upon hydrolysis by the corresponding enzyme,of a detectable color change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase reporter module; forhorseradish peroxidase, 1,2-phenylenediamine, 5-aminosalicylic acid ortolidine are commonly used. Incorporation of a reporter module into asequence encoding a multimodal TRAIL agent can be by any method known tothe skilled artisan, for example by nick translation, primer extension,random oligo priming, by 3′ or 5′ end labeling or by other means (see,for example, Sambrook et al. Molecular Biology: A laboratory Approach,Cold Spring Harbor, N.Y. 1989).

In some embodiments of the aspects described herein, the reporter modulecomprises a variant of luciferase. In some such embodiments, thereporter module comprises an alternative form of Renilla luciferasedesignated RLo (Vesinik et al. Molec Imag Biol 2007, 9:267-77). In otherembodiments, the luciferase can be firefly luciferase (FLuc), Renillaluciferase (Rluc), or Gaussia princeps luciferase (GpLuc). Othervariants of luciferase can be used and are known to those skilled in theart (Greer and Szalay Luminesence 2002, 17:43-74). For example,Arachnocampa (Diptera) luciferases and uses thereof are taught in USPub. No. 20110015095. Also, engineered forms of Renilla luciferase withgreater stability and light emission than the native enzyme can be used(Loening et al., Nature Methods 2007 4:641-3). For example, fireflyluciferase that is chemically modified by covalent linking or is abiotinylated fusion protein can provide near-infrared imagingcapabilities using bioluminescence resonance energy transfer (BRET)(Branchini et al., Bioconjugate Chem 2010 21:2023-2030).

In other embodiments of the aspects described herein, the reportermodule comprises HSV1-TK or its variants or mutants (e.g., HSV1-sr39TK).The two main categories of substrates for TK, uracil nucleosidederivatives labeled with radioactive iodine (e.g., FIAU or radiolabeled2′-fluoro-2′-deoxyarabinofuranosyl-5-ethyl uracil (FEAU)), andacycloguanosine derivatives labeled with radioactive ¹⁸F-Fluorine (e.g.,fluoropenciclovir [FPCV] or9-(4-[¹⁸F]-fluoro-3-hydroxymethylbutyl)-guanine [FHBG]), have beeninvestigated in the last few years as reporter probes for imagingHSV1-tk reporter gene expression. These radiolabeled reporter probes arephosphorylated by TK. If HSV1-TK is expressed intracellularly, thephosphorylation of the substrate will trap it in the cell expressingHSV1-TK. When used in non-pharmacological tracer doses, these substratescan serve as positron emission tomography (PET) or single photonemission computed tomography (SPECT) targeted reporter probes by theiraccumulation in those locations where HSV1-TK is present. Additionally,a mutant version of this gene, HSV1-sr39tk was derived usingsite-directed mutagenesis to obtain an enzyme (HSV1-sr39TK) moreeffective at phosphorylating ganciclovir/penciclovir, and also lessefficient at phosphorylating thymidine, with consequent gain in imagingsignal (Yaghoubi and Gambhir Nat Protoc 2006 1:3069-75; Najjar et al. JNuc Med 2009 50:409-16; Alauddin et al. Curr Topics Med Chem 201010:1617-32; Likar et al. Eur J Nuc Med Mol Imag 2009 36:1273-82;Soghomonyan et al., Nat Protoc 2007 2:416-23, the contents of each ofwhich are herein incorporated by reference in their entireties).

In some embodiments, the reporter module can comprise the Dopamine Type2 Receptor (D2R) PET reporter module which specifically binds3-(2′-[18F]fluoroethyl)spiperone (FESP) and is imaged by PET or SPECT(Aung et al., Nucl Med Commun 2005 26:259-68). In other embodiments, thereporter module can comprise a sequence that binds MRI contrast agents(US Pub. No. 20100322861, the contents of which are herein incorporatedby reference in their entireties). In other embodiments, the reportermodule, can comprise a somatostatin Type 2 receptor (Henze et al., JNucl Med 2001, 42:1053-6) and Sodium/Iodide Symporter (Terrovitis etal., J Am Coll Cardiol 2008, 52:1652-60), the activity of which may bedetected by PET or SPECT. In other embodiments, the reporter module cancomprise (green/red) fluorescent protein and variants thereof, like EGFP(enhanced green fluorescent protein), RFP (red fluorescent protein, likeDsRed or DsRed2), CFP (cyan fluorescent protein), BFP (blue greenfluorescent protein), YFP (yellow fluorescent protein),beta.-galactosidase, or chloramphenicol acetyltransferase, and the like(Ray et al. Semin Nucl Med 2001 31:312-20; Deroose et al. Curr Gene Ther2009 9:212-38; Nair-Gill et al. Immunol Rev 2008 221:214-28; Holmbergand Ahlgren Diabetologia 2008 51:2148-2154). For example, GFP can befrom Aequorea victoria (U.S. Pat. No. 5,491,084). A plasmid encoding theGFP of Aequorea victoria is available from the ATCC Accession No. 87451.Other mutated forms of this GFP including, but not limited to, pRSGFP,EGFP, RFP/DsRed, and EYFP, BFP, YFP, among others, are commerciallyavailable from, inter alia, Clontech Laboratories, Inc. (Palo Alto,Calif.). For example, DsRed2 is also available from ClontechLaboratories, Inc. (Palo Alto, Calif.).

Linker Modules

As described herein, the inventors have further elucidated anddetermined that increased intramolecular spacing between the reporterand the therapeutic TRAIL modules of a multimodal TRAIL agent issurprisingly critical for the effectiveness of the multifunctionalproperties of the multimodal TRAIL agents. As described herein, amultimodal TRAIL module comprising a linker domain having the 7 aminoacids (EASGGPE; SEQ ID NO: 3), as shown in Example 1 and FIG. 1,resulted in much less reporter module expression and TRAIL functionalactivity than a multimodal TRAIL module comprising a linker domainhaving 18 amino acids (GSTGGSGKPGSGEGSTGG; SEQ ID NO: 4). Thus, in someembodiments of the aspects described herein, the multimodal TRAIL agentsfurther comprise a linker domain C-terminal to the reporter module andN-terminal to the therapeutic TRAIL module.

As used herein, a “linker module” refers to a peptide, or a nucleotidesequence encoding such a peptide, of at least 8 amino acids in length.In some embodiments of the aspects described herein, the linker modulecomprises at least 9 amino acids, at least 10 amino acids, at least 11amino acids, at least 12 amino acids, at least 13 amino acids, at least14 amino acids, at least 15 amino acids, at least 16 amino acids, atleast 17 amino acids, at least 18 amino acids, at least 19 amino acids,at least 20 amino acids, at least 21 amino acids, at least 22 aminoacids, at least 23 amino acids, at least 24 amino acids, at least 25amino acids, at least 30 amino acids, at least 35 amino acids, at least40 amino acids, at least 45 amino acids, at least 50 amino acids, atleast 55 amino acids, at least 56 amino acids, at least 60 amino acids,or least 65 amino acids. In some embodiments of the aspects describedherein, a linker module comprises a peptide of 18 amino acids in length.In some embodiments of the aspects described herein, a linker modulecomprises a peptide of at least 8 amino acids in length but less than orequal to 56 amino acids in length, i.e., the length of the spacersequence in the native TRAIL molecule of SEQ ID NO: 1. In someembodiments, the linker molecule comprises the spacer sequence of humanTRAIL, i.e., amino acids 39-94 of SEQ ID NO: 1, or a sequence having atleast 80%, at least 85%, at least 90%, at least 95%, at least 99%identity to amino acids 39-94 of SEQ ID NO: 1.

In some embodiments of the aspects described herein, a linker modulecomprises an amino acid sequence of SEQ ID NO: 4. In some embodiments ofthe aspects described herein, a linker module consists essentially of anamino acid sequence of SEQ ID NO: 4. In some embodiments of the aspectsdescribed herein, a linker module consists of an amino acid sequence ofSEQ ID NO: 4.

Modes of Direct Administration

The multimodal TRAIL agents described herein can be administereddirectly as a pharmaceutical composition to a subject in need thereof byany appropriate route which results in an effective treatment in thesubject. As used herein, the terms “administering,” and “introducing”are used interchangeably and refer to the placement of a multimodalTRAIL agent into a subject by a method or route which results in atleast partial localization of such agents at a desired site, such as acancerous or tumor site or a tumor resection site, such that a desiredeffect(s) is produced.

In some embodiments of the methods described herein, the multimodalTRAIL agent is administered to a subject in need thereof by any mode ofadministration that delivers the agent systemically or to a desiredsurface or target, and can include, but is not limited to, injection,infusion, instillation, and inhalation administration. To the extentthat polypeptide agents can be protected from inactivation in the gut,oral administration forms are also contemplated. “Injection” includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. In someembodiments, the multimodal TRAIL agents for use in the methodsdescribed herein are administered by intravenous infusion or injection.

The phrases “parenteral administration” and “administered parenterally”as used herein, refer to modes of administration other than enteral andtopical administration, usually by injection. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein refer tothe administration of the multimodal TRAIL agent other than directlyinto a target site, tissue, or organ, such as a tumor site, such that itenters the subject's circulatory system and, thus, is subject tometabolism and other like processes.

For the clinical use of the methods described herein, administration ofthe multimodal TRAIL agents can include formulation into pharmaceuticalcompositions or pharmaceutical formulations for parenteraladministration, e.g., intravenous; mucosal, e.g., intranasal; ocular, orother mode of administration. In some embodiments of the methods, themultimodal TRAIL agents described herein can be administered along withany pharmaceutically acceptable carrier compound, material, orcomposition which results in an effective treatment in the subject.Thus, a pharmaceutical formulation for use in the methods describedherein can comprise a multimodal TRAIL agent as described herein incombination with one or more pharmaceutically acceptable ingredients.

The phrase “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio. The phrase “pharmaceutically acceptablecarrier” as used herein means a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent, media, encapsulating material, manufacturing aid(e.g., lubricant, talc magnesium, calcium or zinc stearate, or stericacid), or solvent encapsulating material, involved in maintaining thestability, solubility, or activity of, a multimodal TRAIL agent. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not injurious to the patient.Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) excipients, such as cocoa butterand suppository waxes; (8) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (9)glycols, such as propylene glycol; (10) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol (PEG); (11) esters, such asethyl oleate and ethyl laurate; (12) agar; (13) buffering agents, suchas magnesium hydroxide and aluminum hydroxide; (14) alginic acid; (15)pyrogen-free water; (16) isotonic saline; (17) Ringer's solution; (19)pH buffered solutions; (20) polyesters, polycarbonates and/orpolyanhydrides; (21) bulking agents, such as polypeptides and aminoacids (22) serum components, such as serum albumin, HDL and LDL; (23)C2-C12 alcohols, such as ethanol; and (24) other non-toxic compatiblesubstances employed in pharmaceutical formulations. Release agents,coating agents, preservatives, and antioxidants can also be present inthe formulation. The terms such as “excipient”, “carrier”,“pharmaceutically acceptable carrier” or the like are usedinterchangeably herein.

The multimodal TRAIL agents described herein can be specially formulatedfor administration to a subject in solid, liquid or gel form, includingthose adapted for the following: (1) parenteral administration, forexample, by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution or suspension, orsustained-release formulation; (2) topical application, for example, asa cream, ointment, or a controlled-release patch or spray applied to theskin; (3) intravaginally or intrarectally, for example, as a pessary,cream or foam; (4) ocularly; (5) transdermally; (6) transmucosally; or(79) nasally. Additionally, a multimodal TRAIL agent can be implantedinto a patient or injected using a drug delivery system. See, forexample, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236(1984); Lewis, ed. “Controlled Release of Pesticides andPharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No.3,773,919; and U.S. Pat. No. 3,270,960.

Some further embodiments of the formulations and modes of directadministration of the multimodal TRAIL agents that can be used in themethods described herein are illustrated below.

Parenteral Dosage Forms.

Parenteral dosage forms of the multimodal TRAIL agents can also beadministered to a subject in need thereof, such as a cancer patient, byvarious routes, including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Sinceadministration of parenteral dosage forms typically bypasses thepatient's natural defenses against contaminants, parenteral dosage formsare preferably sterile or capable of being sterilized prior toadministration to a patient. Examples of parenteral dosage formsinclude, but are not limited to, solutions ready for injection, dryproducts ready to be dissolved or suspended in a pharmaceuticallyacceptable vehicle for injection, suspensions ready for injection,controlled-release parenteral dosage forms, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms asdescribed herein are well known to those skilled in the art. Examplesinclude, without limitation: sterile water; water for injection USP;saline solution; glucose solution; aqueous vehicles such as but notlimited to, sodium chloride injection, Ringer's injection, dextroseInjection, dextrose and sodium chloride injection, and lactated Ringer'sinjection; water-miscible vehicles such as, but not limited to, ethylalcohol, polyethylene glycol, and propylene glycol; and non-aqueousvehicles such as, but not limited to, corn oil, cottonseed oil, peanutoil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

Controlled and Delayed Release Dosage Forms.

In some embodiments of the aspects described herein, a multimodal TRAILagent can be administered to a subject by controlled- or delayed-releasemeans. Ideally, the use of an optimally designed controlled-releasepreparation in medical treatment is characterized by a minimum ofdrug/agent substance being employed to cure or control the condition ina minimum amount of time. Advantages of controlled-release formulationsinclude: 1) extended activity of the drug; 2) reduced dosage frequency;3) increased patient compliance; 4) usage of less total drug; 5)reduction in local or systemic side effects; 6) minimization of drugaccumulation; 7) reduction in blood level fluctuations; 8) improvementin efficacy of treatment; 9) reduction of potentiation or loss of drugactivity; and 10) improvement in speed of control of diseases orconditions. (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2(Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-releaseformulations can be used to control a multimodal TRAIL agent's onset ofaction, duration of action, plasma levels within the therapeutic window,and peak blood levels. In particular, controlled- or extended-releasedosage forms or formulations can be used to ensure that the maximumeffectiveness of a multimodal TRAIL agent is achieved while minimizingpotential adverse effects and safety concerns, which can occur both fromunder-dosing a drug (i.e., going below the minimum therapeutic levels)as well as exceeding the toxicity level for the drug.

A variety of known controlled- or extended-release dosage forms,formulations, and devices can be adapted for use with the multimodalTRAIL agents described herein. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548;5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1, each ofwhich is incorporated herein by reference in their entireties. Thesedosage forms can be used to provide slow or controlled-release of one ormore active ingredients using, for example, hydroxypropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmoticsystems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)),multilayer coatings, microparticles, liposomes, or microspheres or acombination thereof to provide the desired release profile in varyingproportions. Additionally, ion exchange materials can be used to prepareimmobilized, adsorbed salt forms of the disclosed agents and thus effectcontrolled delivery of the drug. Examples of specific anion exchangersinclude, but are not limited to, Duolite® A568 and Duolite® AP143(Rohm&Haas, Spring House, Pa. USA).

In some embodiments, a multimodal TRAIL agent for use in the methodsdescribed herein is administered to a subject by sustained release or inpulses. Pulse therapy is not a form of discontinuous administration ofthe same amount of a composition over time, but comprises administrationof the same dose of the composition at a reduced frequency oradministration of reduced doses. Sustained release or pulseadministrations are preferred when the disorder occurs continuously inthe subject, for example where the subject has a chronic disorder suchas cancer. Each pulse dose can be reduced and the total amount of amultimodal TRAIL agent administered over the course of treatment to thepatient is minimized.

The interval between pulses, when necessary, can be determined by one ofordinary skill in the art. Often, the interval between pulses can becalculated by administering another dose of a composition comprising amultimodal TRAIL agent when the composition or the active component ofthe composition is no longer detectable in the subject prior to deliveryof the next pulse. Intervals can also be calculated from the in vivohalf-life of the composition. Intervals can be calculated as greaterthan the in vivo half-life, or 2, 3, 4, 5 and even 10 times greater thecomposition half-life. Various methods and apparatus for pulsingcompositions by infusion or other forms of delivery to the patient aredisclosed in U.S. Pat. Nos. 4,747,825; 4,723,958; 4,948,592; 4,965,251and 5,403,590.

Vectors and Genetic Engineering

Gene therapy or transgene compositions and methods thereof are alsocontemplated for use with the multimodal TRAIL agents described herein.Such methods allow clinicians to introduce a nucleic acid sequenceencoding a multimodal TRAIL agent or component thereof of interestdirectly into a patient (in vivo gene therapy) or into cells isolatedfrom a patient or a donor (ex vivo gene therapy). Therapeutic multimodalTRAIL agents produced by transduced cells after gene therapy can bemaintained at a relatively constant level in, for example, the CNS of asubject, as compared to a protein that is administered directly. Suchsustained production of a multimodal TRAIL agent is particularlyappropriate in the treatment of chronic diseases, such as cancers.Expression can be transient (on the order of hours to weeks) orsustained (weeks to months or longer), depending upon the specificconstruct used and the target tissue or cell type. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA(1995) 92:1292).

Further, regulatable genetic constructs using small molecule inducershave been developed that can be included in vectors to be used in someembodiments of the aspects described herein. (Rivera et al. (1996) Nat.Med. 2:1028-32; No et al. (1996) Proc. Natl. Acad. Sci. USA, 93:3346-51;Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-51; theGeneSwitch® system (Valentis, Inc., Burlingame, Calif.)). These systemsare based on the use of engineered transcription factors the activity ofwhich is controlled by a small molecule drug, and a transgene, theexpression of which is driven by the regulated transcription factor(Rivera et al. (1996) Nat. Med. 2:1028-32; Pollock et al. (2000) Proc.Natl. Acad. Sci. USA 97:13221-26; U.S. Pat. Nos. 6,043,082 and6,649,595; Rivera et al. (1999) Proc. Natl. Acad. Sci. USA 96:8657-62).

In some of the aspects described herein, a nucleic acid sequenceencoding a multimodal TRAIL agent, or any module thereof, is operablylinked to a vector. In general, as used herein, the term “vector” refersto any genetic element, such as a plasmid, phage, transposon, cosmid,chromosome, virus, virion, etc., that is capable of replication whenassociated with the proper control elements and that can transfer genesequences to cells. Thus, the term includes cloning and expressionvehicles, as well as viral vectors. By “recombinant vector” is meant avector that includes a heterologous nucleic acid sequence, or“transgene” that is capable of expression in vivo. It should beunderstood that the vectors described herein can, in some embodiments,be combined with other suitable compositions and therapies. Vectorsuseful for the delivery of a sequence encoding a multimodal TRAIL agentor component thereof can include onr or more regulatory elements (e.g.,promoter, enhancer, etc.) sufficient for expression of the multimodalTRAIL agent or component thereof in the desired target cell or tissue.The regulatory elements can be chosen to provide either constitutive orregulated/inducible expression.

Plasmid-Directed Delivery

A nucleic acid sequence encoding a multimodal TRAIL agent or any modulethereof, can, in some embodiments, be delivered using non-viral,plasmid-based nucleic acid delivery systems, as described in U.S. Pat.Nos. 6,413,942, 6,214,804, 5,580,859, 5,589,466, 5,763,270 and5,693,622, all of which are incorporated herein by reference in theirentireties. Such plasmids comprise the sequence encoding the multimodalTRAIL agent, or a component thereof, operably linked to control elementsthat direct the expression of the multimodal TRAIL agent in a targetcell, and are well known to those of ordinary skill in the art.

In some embodiments, plasmid vectors comprising nucleic acid sequence(s)encoding a multimodal TRAIL agent or any module thereof can be packagedin liposomes prior to delivery to a subject or to cells, as described inU.S. Pat. Nos. 5,580,859, 5,549,127, 5,264,618, 5,703,055, allincorporated herein by reference in their entireties. For a review ofthe use of liposomes as carriers for delivery of nucleic acids, see, Hugand Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger et al.(1983) in Methods of Enzymology Vol. 101, pp. 512-27; de Lima et al.(2003) Current Medicinal Chemistry, Volume 10(14): 1221-31. The DNA canalso be delivered in cochleate lipid compositions similar to thosedescribed by Papahadjopoulos et al. (1975) Biochem. Biophys. Acta.394:483-491. See also U.S. Pat. Nos. 4,663,161 and 4,871,488,incorporated herein by reference in their entireties.

Biolistic delivery systems employing particulate carriers such as goldand tungsten can also be used to deliver nucleic acid sequence encodinga multimodal TRAIL agent, or any module thereof. See, e.g., U.S. Pat.Nos. 4,945,050, 5,036,006, 5,100,792, 5,179,022, 5,371,015, and5,478,744, all incorporated herein by reference in their entireties.

A wide variety of other methods can be used to deliver the vectorscomprising nucleic acid sequence(s) encoding a multimodal TRAIL agent orany module thereof. Such methods include DEAE dextran-mediatedtransfection, calcium phosphate precipitation, polylysine- orpolyornithine-mediated transfection, or precipitation using otherinsoluble inorganic salts, such as strontium phosphate, aluminumsilicates including bentonite and kaolin, chromic oxide, magnesiumsilicate, talc, and the like. Other useful methods of transfectioninclude electroporation, sonoporation, protoplast fusion, peptoiddelivery, or microinjection. See, e.g., Sambrook et al (1989) MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York,for a discussion of techniques for transforming cells of interest; andFelgner, P. L. (1990) Advanced Drug Delivery Reviews 5:163-87, for areview of delivery systems useful for gene transfer. Exemplary methodsof delivering DNA using electroporation are described in U.S. Pat. Nos.6,132,419; 6,451,002, 6,418,341, 6,233,483, U.S. Patent Publication No.2002/0146831, and International Publication No. WO/0045823, all of whichare incorporated herein by reference in their entireties.

In other embodiments of the aspects described herein, plasmid vectorsvectors comprising nucleic acid sequence(s) encoding a multimodal TRAILagent or any module thereof can also be introduced directly into the CNSby intrathecal (IT) injection, as described herein in greater detailwith regard to protein administration. Plasmid DNA can be complexed withcationic agents such as polyethyleneimine (PEI) or Lipofectamine 2000 tofacilitate uptake.

Retroviral Delivery

Retroviruses, such as lentiviruses, provide another convenient platformfor delivery of nucleic acid sequences encoding a multimodal TRAIL agentof interest. A selected nucleic acid sequence can be inserted into avector and packaged in retroviral particles using techniques known inthe art. These retroviral vectors contain the components necessary forthe correct packaging of the viral genome and integration into the hostcell DNA. The recombinant virus can then be isolated and delivered tocells of the subject either in vivo or ex vivo. The nucleic acidsequences encoding a multimodal TRAIL agent or module thereof are clonedinto one or more vectors, which facilitates delivery of the nucleic acidinto a patient. Retroviral systems are described in, for example, U.S.Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-90;Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991)Virology 180:849-52; Miller et al., Meth. Enzymol. 217:581-599 (1993);Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-37; Boris-Lawrieand Temin (1993) Curr. Opin. Genet. Develop. 3:102-09. Greater detailabout retroviral vectors can be found, for example, in Boesen et al.,Biotherapy 6:291-302 (1994), which describes the use of a retroviralvector to deliver the mdr1 gene to hematopoietic stem cells in order tomake the stem cells more resistant to chemotherapy. Other referencesillustrating the use of retroviral vectors in gene therapy include:Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141(1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel.3:110-114 (1993), the contents of each of which are herein incorporatedby reference in their entireties.

In some embodiments of the aspects described herein, a lentiviral systemis used to deliver a nucleic acid sequence encoding a multimodal TRAILagent of interest. Lentiviral vectors contemplated for use include, forexample, the HIV based vectors described in U.S. Pat. Nos. 6,143,520;5,665,557; and 5,981,276, the contents of which are herein incorporatedby reference in their entireties.

Adenoviral Delivery

In some embodiments, a nucleotide sequence encoding a multimodal TRAILagent of interest or module thereof is inserted into an adenovirus-basedexpression vector. Unlike retroviruses, which integrate into the hostgenome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-74; Bett et al. (1993) J. Virol. 67:5911-21;Mittereder et al. (1994) Human Gene Therapy 5:717-29; Seth et al. (1994)J. Virol. 68:933-40; Barr et al. (1994) Gene Therapy 1:51-58; Berkner,K. L. (1988) BioTechniques 6:616-29; and Rich et al. (1993) Human GeneTherapy 4:461-76). Adenoviral vectors have several advantages in genetherapy. They infect a wide variety of cells, have a broad host-range,exhibit high efficiencies of infectivity, direct expression ofheterologous sequences at high levels, and achieve long-term expressionof those sequences in vivo. The virus is fully infective as a cell-freevirion so injection of producer cell lines is not necessary. With regardto safety, adenovirus is not associated with severe human pathology, andthe recombinant vectors derived from the virus can be renderedreplication defective by deletions in the early-region 1 (“E1”) of theviral genome. Adenovirus can also be produced in large quantities withrelative ease. For all these reasons vectors derived from humanadenoviruses, in which at least the E1 region has been deleted andreplaced by a gene of interest, have been used extensively for genetherapy experiments in the pre-clinical and clinical phase.

Adenoviral vectors for use with the compositions and methods describedherein can be derived from any of the various adenoviral serotypes,including, without limitation, any of the over 40 serotype strains ofadenovirus, such as serotypes 2, 5, 12, 40, and 41. The adenoviralvectors used herein are replication-deficient and contain the sequenceof interest under the control of a suitable promoter, such as any of thepromoters discussed below with reference to adeno-associated virus. Forexample, U.S. Pat. No. 6,048,551, incorporated herein by reference inits entirety, describes replication-deficient adenoviral vectors thatinclude a human gene under the control of the Rous Sarcoma Virus (RSV)promoter. Other recombinant adenoviruses of various serotypes, andcomprising different promoter systems, can be created by those skilledin the art. See, e.g., U.S. Pat. No. 6,306,652, incorporated herein byreference in its entirety.

Other useful adenovirus-based vectors for delivery of nucleic acidsequence encoding a multimodal TRAIL agent of interest or module thereofinclude, but are not limited to: “minimal” adenovirus vectors asdescribed in U.S. Pat. No. 6,306,652, which retain at least a portion ofthe viral genome required for encapsidation (the encapsidation signal),as well as at least one copy of at least a functional part or aderivative of the ITR; and the “gutless” (helper-dependent) adenovirusin which the vast majority of the viral genome has been removed andwhich produce essentially no viral proteins, thus allowing gene therapyto persist for over a year after a single administration (Wu et al.(2001) Anesthes. 94:1119-32; Parks (2000) Clin. Genet. 58:1-11; Tsai etal. (2000) Curr. Opin. Mol. Ther. 2:515-23).

Adeno Associated Virus (AAV) Delivery

In some embodiments of the compositions and methods described herein, anucleotide sequence encoding a multimodal TRAIL agent of interest isinserted into an adeno-associated virus-based expression vector. AAV isa parvovirus which belongs to the genus Dependovirus and has severalfeatures not found in other viruses. AAV can infect a wide range of hostcells, including non-dividing cells. AAV can infect cells from differentspecies. AAV has not been associated with any human or animal diseaseand does not appear to alter the biological properties of the host cellupon integration. Indeed, it is estimated that 80-85% of the humanpopulation has been exposed to the virus. Finally, AAV is stable at awide range of physical and chemical conditions, facilitating production,storage and transportation.

AAV is a helper-dependent virus; that is, it requires co-infection witha helper virus (e.g., adenovirus, herpesvirus or vaccinia) in order toform AAV virions in the wild. In the absence of co-infection with ahelper virus, AAV establishes a latent state in which the viral genomeinserts into a host cell chromosome, but infectious virions are notproduced. Subsequent infection by a helper virus rescues the integratedgenome, allowing it to replicate and package its genome into infectiousAAV virions. While AAV can infect cells from different species, thehelper virus must be of the same species as the host cell. Thus, forexample, human AAV will replicate in canine cells co-infected with acanine adenovirus.

Adeno-associated virus (AAV) has been used with success in gene therapy.AAV has been engineered to deliver genes of interest by deleting theinternal nonrepeating portion of the AAV genome (i.e., the rep and capgenes) and inserting a heterologous sequence (in this case, the sequenceencoding the multimodal TRAIL agent) between the ITRs. The heterologoussequence is typically functionally linked to a heterologous promoter(constitutive, cell-specific, or inducible) capable of drivingexpression in the patient's target cells under appropriate conditions.

Recombinant AAV virions comprising a nucleic acid sequence encoding amultimodal TRAIL agent of interest can be produced using a variety ofart-recognized techniques, as described in U.S. Pat. Nos. 5,139,941;5,622,856; 5,139,941; 6,001,650; and 6,004,797, the contents of each ofwhich are incorporated by reference herein in their entireties. Vectorsand cell lines necessary for preparing helper virus-free rAAV stocks arecommercially available as the AAV Helper-Free System (Catalog No.240071) (Stratagene, La Jolla, Calif.).

Other Viral Vectors for Delivery

Additional viral vectors useful for delivering nucleic acid moleculesencoding a multimodal TRAIL agent of interest include those derived fromthe pox family of viruses, including vaccinia virus and avian poxvirus.Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can be used to deliver the genes. The use of avipox vectors in human andother mammalian species is advantageous with regard to safety becausemembers of the avipox genus can only productively replicate insusceptible avian species. Methods for producing recombinantavipoxviruses are known in the art and employ genetic recombination,see, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Molecular conjugate vectors, such as the adenovirus chimeric vectors,can also be used for delivery of sequence encoding a multimodal TRAILagent or component thereof (Michael et al. (1993) J. Biol. Chem.268:6866-69 and Wagner et al. (1992) Proc. Natl. Acad. Sci. USA89:6099-6103). Members of the Alphavirus genus, for example the Sindbisand Semliki Forest viruses, can also be used as viral vectors fordelivering a nucleic acid sequence encoding a multimodal TRAIL agent ofinterest (See, e.g., Dubensky et al. (1996) J. Virol. 70:508-19; WO95/07995; WO 96/17072).

Cell Types

Essentially any cell type can be engineered with a sequence encoding themultimodal TRAIL agents, as described herein, for use in cellulartherapies. Thus, differentiated somatic cells and stem cells, as well ascells of a cell line, can be engineered to express, using any methodknown to one of skill in the art, a desired multimodal TRAIL agent. Insome embodiments of the aspects described herein, a cell can betransduced with a delivery vector comprising a nucleic acid sequenceencoding a multimodal TRAIL agent or module thereof. In otherembodiments of the compositions and methods described herein, a cell canbe transfected with a nucleic acid sequence encoding a multimodal TRAILagent. Provided herein are exemplary stem cells, somatic cells, and cellline sources useful with the methods and compositions described herein.However, the description herein is not meant to be limiting and any cellknown or used in the art can be genetically modified or engineered toexpress and secrete a multimodal TRAIL agent. In some embodiments, thecells to be engineered can be from an autologous, i.e., from the samesubject, or from one or more heterologous sources.

Stem Cells

Stem cells are undifferentiated cells defined by their ability at thesingle cell level to both self-renew and differentiate to produceprogeny cells, including self-renewing progenitors, non-renewingprogenitors, and terminally differentiated cells. Stem cells, dependingon their level of differentiation, are also characterized by theirability to differentiate in vitro into functional cells of various celllineages from multiple germ layers (endoderm, mesoderm and ectoderm), aswell as to give rise to tissues of multiple germ layers followingtransplantation and to contribute substantially to most, if not all,tissues following injection into blastocysts. (See, e.g., Potten et al.,Development 110: 1001 (1990); U.S. Pat. Nos. 5,750,376, 5,851,832,5,753,506, 5,589,376, 5,824,489, 5,654,183, 5,693,482, 5,672,499, and5,849,553, all herein incorporated in their entireties by reference).

The stem cells for use with the compositions and methods comprisingmultimodal TRAIL agents described herein can be naturally occurring stemcells or “induced” stem cells, such as “induced pluripotent stem cells”(iPS cells) generated using any method or composition known to one ofskill in the art. Stem cells can be obtained or generated from anymammalian species, e.g. human, primate, equine, bovine, porcine, canine,feline, rodent, e.g. mice, rats, hamster, etc. In some embodiments ofthe aspects described herein, a stem cell is a human stem cell.

Stem cells are classified by their developmental potential as: (1)totipotent, meaning able to give rise to all embryonic andextraembryonic cell types; (2) pluripotent, meaning able to give rise toall embryonic cell types; (3) multipotent, meaning able to give rise toa subset of cell lineages, but all within a particular tissue, organ, orphysiological system (for example, hematopoietic stem cells (HSC) canproduce progeny that include HSC (self-renewal), blood cell restrictedoligopotent progenitors and the cell types and elements (e.g.,platelets) that are normal components of the blood); (4) oligopotent,meaning able to give rise to a more restricted subset of cell lineagesthan multipotent stem cells; and (5) unipotent, meaning able to giverise to a single cell lineage (e.g., spermatogenic stem cells).

Stem cells of interest for use in the compositions and methods describedherein include embryonic cells of various types, exemplified by humanembryonic stem (hES) cells, described by Thomson et al. (1998) Science282:1145; embryonic stem cells from other primates, such as Rhesus stemcells (Thomson et al. (1995) Proc. Natl. Acad. Sci USA 92:7844);marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); andhuman embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad.Sci. USA 95:13726, 1998). Cells derived from embryonic sources caninclude embryonic stem cells or stem cell lines obtained from a stemcell bank or other recognized depository institution.

In some embodiments of the aspects described herein, a cell engineeredto express or secrete a multimodal TRAIL agent is an adult or somaticstem cell. Adult stem cells are generally limited to differentiatinginto different cell types of their tissue of origin. However, if thestarting stem cells are derived from the inner cell mass of the embryo,they can generate many cell types of the body derived from all threeembryonic cell types: endoderm, mesoderm and ectoderm. Stem cells withthis property are said to be “pluripotent.” Embryonic stem cells are onekind of pluripotent stem cell. Thus, pluripotent embryonic stem cellscan be differentiated into many specific cell types. Since the embryo isa potential source of all types of precursor cells, it is possible todifferentiate, for example, engineered embryonic stem cells into otherlineages by providing the appropriate signals, such as the expression ofproteins, using any method known to one of skill in the art, toembryonic stem cells.

Somatic or adult stem cells have major advantages, for example, as usingsomatic stem cells allows a patient's own cells to be expanded inculture and then re-introduced into the patient. Natural somatic stemcells have been isolated from a wide variety of adult tissues includingblood, bone marrow, brain, olfactory epithelium, skin, pancreas,skeletal muscle, and cardiac muscle. Each of these somatic stem cellscan be characterized based on gene expression, factor responsiveness,and morphology in culture. Exemplary naturally occurring somatic stemcells include, but are not limited to, neural stem cells, neural creststem cells, mesenchymal stem cells, hematopoietic stem cells, andpancreatic stem cells. In addition, iPS cells generated from a patientprovide a source of cells that can be engineered to express a multimodalTRAIL agent, expanded, and re-introduced to the patient, before or afterstimulation to differentiate to a desired lineage or phenotype, such asa neural stem cell. In some embodiments of the aspects described herein,a somatic stem cell engineered to express a multimodal TRAIL agent is aneural stem cell. In some embodiments of the aspects described herein, asomatic stem cell engineered to express a multimodal TRAIL agent is amesenchymal stem cell. In some embodiments of the aspects describedherein, a somatic stem cell engineered to express a multimodal TRAILagent is an iPS cell differentiated into a neural stem cell. In someembodiments of the aspects described herein, a somatic stem cellengineered to express a multimodal TRAIL agent is an iPS celldifferentiated into a mesenchymal stem cell.

Cord blood cells are used as a source of transplantable stem andprogenitor cells and as a source of marrow repopulating cells for thetreatment of malignant diseases (e.g, acute lymphoid leukemia, acutemyeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome,and nueroblastoma) and non-malignant diseases such as Fanconi's anemiaand aplastic anemia (Kohli-Kumar et al., 1993 Br. J. Haematol.85:419-422; Wagner et al., 1992 Blood 79; 1874-1881; Lu et al., 1996Crit. Rev. Oncol. Hematol 22:61-78; Lu et al., 1995 Cell Transplantation4:493-503). Accordingly, in some aspects, cells to be engineered tosecrete or express a multimodal TRAIL agent can also be derived fromhuman umbilical cord blood cells (HUCBC), which are recognized as a richsource of hematopoietic and mesenchymal stem cells (Broxmeyer et al.,1992 Proc. Natl. Acad. Sci. USA 89:4109-4113). One advantage of HUCBCfor use with the methods and compositions described herein is theimmature immunity of these cells, which is very similar to fetal cells,and thus significantly reduces the risk for rejection by the host(Taylor & Bryson, 1985 J. Immunol. 134:1493-1497).

In some embodiments of the aspects described herein, iPS cells areengineered to express or secrete the multimodal TRAIL agents describedherein. In some embodiments of the aspects described herein, iPS cellsare engineered to express or secrete the multimodal TRAIL agents priorto being differentiated into another desired cell type. In someembodiments of the aspects described herein, iPS cells are engineered toexpress or secrete the multimodal TRAIL agents after differentiationinto another desired cell type.

In other embodiments of the aspects described herein, cancer stem cellscan be engineered to express or secrete the multimodal TRAIL agentsdescribed herein. It has been recently discovered that stem-like cellsare present in some human tumors and, while representing a smallminority of the total cellular mass of the tumor, are the subpopulationof tumor cells responsible for growth of the tumor. In contrast tonormal stem cells, “tumor stem cells” or “cancer stem cells” are definedas cells that can undergo self-renewal, as well as abnormalproliferation and differentiation to form a tumor. Functional featuresof tumor stem cells are that they are tumorigenic; they can give rise toadditional tumorigenic cells by self-renewal; and they can give rise tonon-tumorigenic tumor cells. The developmental origin of tumor stemcells can vary among different types of cancers. It is believed, withoutwishing to be bound or limited by theory, that tumor stem cells canarise either as a result of genetic damage that deregulates normalmechanisms of proliferation and differentiation of stem cells (Lapidotet al., Nature 367(6464): 645-8 (1994)), or by the dysregulatedproliferation of populations of cells that acquire stem-like properties.

Neural Stem Cells

Recent studies have shown that intracranially or intravenously injectedneural stem cells (NSCs) or neural precursor cells migrate towardsinjured or pathological central nervous system (CNS) sites. Thischemotropic property of NSCs has been utilized for cell-based therapiesto treat diverse neurological diseases as described herein and in T.Bagci-Onder et al., Cancer Research 2011, 71:154-163; Hingtgen S. etal., Stem Cells 2010, 28(4):832-41; Hingtgen S. et al., Mol Cancer Ther.2008, 7(11): 3575-85; Brustle O. et al., 6 Current Opinion inNeurobiology. 688 (1996); Flax J. D., et al., 16 Nature Biotechnology.1033. (1998); Kim S. U., 24. Neuropathology. 159 (2004); Lindvall O etal., 10 (suppl) Nature Medicine. S42 (2004); Goldman S., 7. NatureBiotechnology. 862 (2005); Muller F. et al., 7 Nature ReviewsNeuroscience. 75 (2006); Lee, J. P., et al. 13 Nature Medicine 439(2007), and Kim S. U. et al., 87 Journal of Neuroscience Research 2183(2009), the contents of each of which are herein incorporated in theirentireties by reference.

Administration of delivery vectors can be performed intracranially orextracranially using known techniques. Stem cells, such as neural stemcells, have been shown to cross the blood-brain barrier and home towardsinjury in brain. Thus, for example stem cells engineered to produce thesecreted multimodal TRAIL agents described herein can be administeredintravenously and are expected to reach desired areas of the brain, suchas the site of a glioblastoma. Further, and importantly from adiagnostic aspect, as the multimodal TRAIL agents comprise a reportermodule, delivery of the cells and agents to a desired area can bevisualized.

Accordingly, in some embodiments of the compositions and methodsdescribed herein, a pharmaceutically acceptable composition comprising aneural stem cell and a multimodal TRAIL agent can be administered to asubject. In some such embodiments, the neural stem cell is geneticallyengineered to express or secrete a multimodal TRAIL agent. Because NSCscan be engineered to package and release replication-defectiveretroviral particles or replication-conditional herpes virus vectorswhich, in turn, can serve as vectors for the transfer of sequences toCNS cells, neural progenitor/stem cells can serve to magnify theefficacy of viral-mediated gene delivery to large regions in the brain.In some such embodiments, the neural stem cell can comprise a vectorencoding a multimodal TRAIL agent. Additional vectors that can be usedin the embodiments described herein include herpes simplex virusvectors, SV 40 vectors, polyoma virus vectors, papilloma virus vectors,picarnovirus vectors, vaccinia virus vectors, and a helper-dependent orgutless adenovirus. In one embodiment, the vector can be a lentivirus.Methods for preparing genetically engineered neural stem cells andcompositions thereof for therapeutic treatment have been described inU.S. Pat. No. 7,393,526 and U.S. Pat. No. 7,655,224, the contents ofwhich are incorporated herein by reference in their entirety.

In various embodiments of the compositions and methods described herein,the neural stem cells that can be used include, but are not limited to,human neural stem cells, mouse neural stem cells HSN-1 cells, fetal pigcells and neural crest cells, bone marrow derived neural stem cells, andhNT cells. HSN-1 cells can be prepared, for example, as described in,e.g., Ronnett et al. (Science 248, 603-605, 1990). The preparation ofneural crest cells in described in U.S. Pat. No. 5,654,183. hNT cellscan be prepared as described in, e.g, Konubu et al. (Cell Transplant 7,549-558, 1998). In some embodiments of the compositions and methodsdescribed herein, the neural stem cells that can be used are neural stemcells derived or differentiated from a precursor stem cell, such as ahuman embryonic stem cell or an induced pluripotent stem (iPS) cell.Such neural stem cells can be generated from or differentiated fromhuman embryonic stem cells, using, for example, compositions and methodsdescribed in Nature Biotechnology 27, 275-280 (2009), “Highly efficientneural conversion of human ES and iPS cells by dual inhibition of SMADsignaling,” the contents of which are herein incorporated by referencein their entireties. Such neural stem cells can be generated from ordifferentiated from iPS cells, using, for example, the compositions andmethods described in US Patent Publication US 2010/0021437 A1, “NEURALSTEM CELLS DERIVED FROM INDUCED PLURIPOTENT STEM CELLS,” the contents ofwhich are herein incorporated by reference in their entireties.

Accordingly, as used herein, “neural stem cells” refers to a subset ofpluripotent cells which have partially differentiated along a neuralcell pathway and express some neural markers including, for example,nestin. Neural stem cells can differentiate into neurons or glial cells(e.g., astrocytes and oligodendrocytes). Thus, “neural stem cellsderived or differentiated from iPS cells” refers to cells that arepluripotent but have partially differentiated along a neural cellpathway (i.e., express some neural cell markers), and themselves are theresult of in vitro or in vivo differentiation iPS cells.

Neural selection factors that can be used to differentiate pluripotentstem cells, such as embryonic stem cells or iPS cells into neural stemcells, include, for example, sonic hedgehog (SHUT), fibroblast growthfactor-2 (FGF-2), and fibroblast growth factor-8 (FGF-8), which can beused alone or in pairwise combination, or all three factors may be usedtogether. In some embodiments, iPS cells are cultured in the presence ofat least SHH and FGF-8. In other embodiments, FGF-2 is omitted.Preferably, the neural stem cells derived from iPS cells express nestin.In some embodiments, the pluripotent stem cells are cultured in thepresence of the one or more neural selection factors for 4, 5, 6, 7, 8,9, 10, 12, 14, 16, 18, 20 days or more. Preferably, the population ofneural stem cells is characterized in that at least 50%, at least 75%,at least 85%, at least 90%, at least 95%, or at least 99% of the cellsof the population expresses nestin. Preferably, the nestin-expressingcells further express at least one of En-1, Pitx3, and Nurr-1. In otherembodiments, the population of neural stem cells has been depleted of atleast 50%, 75%, 85%, 95%, or 99% of the cells expressing surface markersof immature embryonic stem cells including, for example, SSEA-1, SSEA-3,SSEA-4, Tra-1-81, and Tra-1-60. Preferably, the population of neuralstem cells contains less than 10%, less than 5%, less than 2.5%, lessthan 1%, or less than 0.1% of cells that express the selected marker(e.g., SSEA-4).

Somatic Cells

In some embodiments, the cells engineered to express or secrete themultimodal TRAIL agents described herein are primary somatic cells. Somenon-limiting examples of primary cells include, but are not limited to,fibroblast, epithelial, endothelial, neuronal, adipose, cardiac,skeletal muscle, immune cells, hepatic, splenic, lung, circulating bloodcells, gastrointestinal, renal, bone marrow, and pancreatic cells. Thecell can be a primary cell isolated from any somatic tissue including,but not limited to, brain, liver, lung, gut, stomach, intestine, fat,muscle, uterus, skin, spleen, endocrine organ, bone, etc. The term“somatic cell” further encompasses primary cells grown in culture,provided that the somatic cells are not immortalized.

Cell Lines

In some embodiments, the cells engineered to express or secrete themultimodal TRAIL agents described herein described herein comprise cellsof a cell line.

Exemplary human cell lines include, but are not limited to, 293T(embryonic kidney), BT-549 (breast), DMS 114 (small cell lung), DU145(prostate), HT-1080 (fibrosarcoma), HEK 293 (embryonic kidney), HeLa(cervical carcinoma), HepG2 (hepatocellular carcinoma), HL-60(TB)(leukemia), HS 578T (breast), HT-29 (colon adenocarcinoma), Jurkat (Tlymphocyte), M14 (melanoma), MCF7 (mammary), MDA-MB-453 (mammaryepithelial), PERC6® (E1-transformed embryonal retina), RXF 393 (renal),SF-268 (CNS), SF-295 (CNS), THP-1 (monocyte-derived macrophages), TK-10(renal), U293 (kidney), UACC-257 (melanoma), and XF 498 (CNS).

Examples of non-human primate cell lines useful in the compositions andmethods provided herein include, but are not limited to, monkey kidney(CVI-76) cells, African green monkey kidney (VERO-76) cells, greenmonkey fibroblast (Cos-1) cells, and monkey kidney (CVI) cellstransformed by SV40 (Cos-7). Additional mammalian cell lines are knownto those of ordinary skill in the art and are catalogued at the AmericanType Culture Collection catalog (ATCC®, Mamassas, Va.).

Examples of rodent cell lines useful in the compositions and methodsprovided herein include, but are not limited to, mouse Sertoli (TM4)cells, mouse mammary tumor (MMT) cells, rat hepatoma (HTC) cells, mousemyeloma (NSO) cells, murine hybridoma (Sp2/0) cells, mouse thymoma (EL4)cells, Chinese Hamster Ovary (CHO) cells and CHO cell derivatives,murine embryonic (NIH/3T3, 3T3 Ll) cells, rat myocardial (H9c2) cells,mouse myoblast (C2C12) cells, and mouse kidney (miMCD-3) cells.

Cellular Therapies and Cellular Administration

The compositions and methods comprising multimodal TRAIL agents areparticularly useful in patients in need of cellular therapies.Accordingly, various aspects and embodiments of the methods andcompositions described herein involve administration of an effectiveamount of a cell or a population of cells, generated using any of thecompositions comprising a multimodal TRAIL agent described herein, orengineered to express a multimodal TRAIL agent as described herein, toan individual or subject in need of a cellular therapy. The cell orpopulation of cells being administered can be an autologous population,or be derived from one or more heterologous sources. The cell can be,for example, a stem cell, such as a lineage-restricted progenitor cell,multipotent cell, or an oligopotent cell, or a fully or partiallydifferentiated progeny of a stem cell. In some embodiments, cellsengineered to secrete a multimodal TRAIL agent can be introduced via ascaffold or encapsulated in a biodegradeable extracellular matrix toenhance retention and release of secreted multimodal TRAIL agents in asubject in need thereof.

A variety of means for administering cells to subjects are known tothose of skill in the art. Such methods can include systemic injection,for example, i.v. injection, or implantation of cells into a target sitein a subject, such as a surgical site. Cells can be inserted into adelivery device which facilitates introduction by injection orimplantation into the subject. Such delivery devices can include tubes,e.g., catheters, for injecting cells and fluids into the body of arecipient subject. In some embodiments, the tubes additionally have aneedle, e.g., through which the cells can be introduced into the subjectat a desired location. The cells can be prepared for delivery in avariety of different forms. For example, cells can be suspended in asolution or gel or embedded in a support matrix when contained in such adelivery device. Cells can be mixed with a pharmaceutically acceptablecarrier or diluent in which the cells remain viable.

Pharmaceutically acceptable carriers and diluents include saline,aqueous buffer solutions, solvents and/or dispersion media. The use ofsuch carriers and diluents is well known in the art. The solution ispreferably sterile and fluid. Preferably, prior to the introduction ofcells as described herein, the solution is stable under the conditionsof manufacture and storage and preserved against the contaminatingaction of microorganisms such as bacteria and fungi through the use ofagents such as parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like.

It is preferred that the mode of cell administration is relativelynon-invasive, for example by intravenous injection, pulmonary deliverythrough inhalation, topical, or intranasal administration. However, theroute of cell administration will depend on the tissue to be treated andcan include implantation. Methods for cell delivery are known to thoseof skill in the art and can be extrapolated by one skilled in the art ofmedicine for use with the methods and compositions described herein.

In some embodiments of the methods described herein, a cell orpopulation of cells engineered to express a multimodal TRAIL agent isdirectly placed or administered to a surgical site, such as a surgicalresection site. By placing the cell or population of cells engineered toexpress a multimodal TRAIL agent at a surgical site, enhanced clearanceof the target cancer cells can be achieved, as demonstrated herein. Suchdirect administration to a surgical site can include administration of asuspension of engineered cells, or encapsulation of engineered cells atthe surgical site, such as, as described herein in Example 2.

In some embodiments of the methods described herein, a cell orpopulation of cells engineered to express a multimodal TRAIL areadministered to a surgical site or lesion (e.g., cancer) site byintraparenchymal (e.g., intracerebral) grafting of the cell or cellpopulations into the surgical or lesioned region. The cells engineeredto express a multimodal TRAIL can be delivered to a specific site bystereotaxic injection. Conventional techniques for grafting aredescribed, for example, in Bjorklund et al. (Neural Grafting in theMammalian CNS, eds. Elsevier, pp 169-178, 1985), Leksell et al. (ActaNeurochir., 52:1-7, 1980) and Leksedl et al. (J. Neurosturg.,66:626-629, 1987).

In some embodiments, administration of engineered cells into selectedregions of a patient's brain can be made by drilling a hole and piercingthe dura to permit the needle of a microsyringe to be inserted.Alternatively, in other embodiments, the cells can be injected into thebrain ventricles or intrathecally into a spinal cord region.

Direct injection techniques for cell administration can also be used tostimulate transmigration of cells through the entire vasculature, or tothe vasculature of a particular organ, such as for example liver, orkidney or any other organ. This includes non-specific targeting of thevasculature. One can target any organ by selecting a specific injectionsite, e.g., a liver portal vein. Alternatively, the injection can beperformed systemically into any vein in the body. In another example,compositions comprising neural stem cells or precursor cells engineeredto secrete a multimodal TRAIL agent can be transplanted directly intoparenchymal or intrathecal sites of the central nervous system,according to the disease being treated, such as for example, the site ofa glioblastoma. Grafts can be done using single cell suspensions orsmall aggregates at a density of 25,000-500,000 cells per mL (U.S. Pat.No. 5,968,829, the contents of which are herein incorporated in theirentireties by reference).

If so desired, a mammal or subject can be pre-treated with an agent, forexample an agent is administered to enhance cell targeting to a tissue(e.g., a homing factor) and can be placed at that site to encouragecells to target the desired tissue. For example, direct injection ofhoming factors into a tissue can be performed prior to systemic deliveryof ligand-targeted cells.

Scaffolds and Encapsulation of Cells

It is further contemplated that, in some embodiments of these aspects,cells engineered to express the multimodal TRAIL agents describedherein, can not only be administered to a subject in need as cells insuspension, but also as cells populating a matrix, scaffold, or othersupport, to enhance retention of cells and delivery of the multimodalTRAIL agents at a site. Encapsulation of stem cells has shown to permitenhanced delivery of engineered stem cells, as described in, forexample, Compte M. et al., Stem Cells 2009, 27(3):753-760, the contentsof which are herein incorporated in their entireties by reference, andas demonstrated herein in the Examples (see, for example, FIGS. 13A-13G,and 17A-17L).

In some embodiments, a “support” refers to any suitable carrier materialto which cells, such as engineered neural stem cells expressing amultimodal TRAIL agent described herein, are able to attach themselvesor adhere, and can be used in order to form a corresponding cellcomposite, e.g. an artificial tissue. In some embodiments, a matrix orcarrier material, respectively, is present already in athree-dimensional form desired for later application.

In some such embodiments, a matrix or a scaffold comprises a“biocompatible substrate” that can be used as a material that issuitable for implantation into a subject onto which a cell populationcan be deposited. A biocompatible substrate does not cause toxic orinjurious effects once implanted in the subject. The biocompatiblesubstrate can provide the supportive framework that allows cells toattach to it, and grow on it. Cultured populations of cells can then begrown on the biocompatible substrate, which provides the appropriateinterstitial distances required for cell-cell interaction.

A matrix, structure, or scaffold can be used to aid in furthercontrolling and directing a cell or population of cells expressing orsecreting a multimodal TRAIL agent described herein. A matrix orscaffold can be designed or selected to provide environmental cues tocontrol and direct the migration of cells to a site of injury ordisease. A structure or scaffold can be engineered from a nanometer tomicrometer to millimeter to macroscopic length, and can further compriseor be based on factors such as, but not limited to, material mechanicalproperties, material solubility, spatial patterning of bioactivecompounds, spatial patterning of topological features, soluble bioactivecompounds, mechanical perturbation (cyclical or static strain, stress,shear, etc. . . . ), electrical stimulation, and thermal perturbation.

A scaffold can be in any desired geometric conformation, for example, aflat sheet, a spiral, a cone, a v-like structure and the like. Ascaffold can be shaped into, e.g., a heart valve, vessel (tubular),planar construct or any other suitable shape. Such scaffold constructsare known in the art (see, e.g., WO02/035992, U.S. Pat. Nos. 6,479,064,6,461,628, the contents of which are herein incorporated in theirentireties by reference). In some embodiments, after culturing the cellson the scaffold, the scaffold is removed (e.g., bioabsorbed orphysically removed), and the cells maintain substantially the sameconformation as the scaffold, such that, for example, if the scaffoldwas spiral shaped, the cells form a 3D-engineered tissue that is spiralshaped.

Biopolymer structures can be generated by providing a transitionalpolymer on a substrate; depositing a biopolymer on the transitionalpolymer; shaping the biopolymer into a structure having a selectedpattern on the transitional polymer (poly(N-Isopropylacrylamide); andreleasing the biopolymer from the transitional polymer with thebiopolymer's structure and integrity intact. A biopolymer can beselected from a natural or synthetic extracellular matrix (ECM) protein,growth factor, lipid, fatty acid, steroid, sugar and other biologicallyactive carbohydrates, a biologically derived homopolymer, nucleic acids,hormone, enzyme, pharmaceutical composition, cell surface ligand andreceptor, cytoskeletal filament, motor protein, silks, polyprotein(e.g., poly(lysine)) or any combination thereof.

The biopolymers used in the generation of the matrices and scaffolds forthe embodiments directed to cellular therapies using multimodal TRAILagents described herein include, but are not limited to, a)extracellular matrix proteins to direct cell adhesion and function(e.g., collagen, fibronectin, laminin, etc.); (b) growth factors todirect cell function specific to cell type (e.g., nerve growth factor,bone morphogenic proteins, vascular endothelial growth factor, etc.);(c) lipids, fatty acids and steroids (e.g., glycerides, non-glycerides,saturated and unsaturated fatty acids, cholesterol, corticosteroids, sexsteroids, etc.); (d) sugars and other biologically active carbohydrates(e.g., monosaccharides, oligosaccharides, sucrose, glucose, glycogen,etc.); (e) combinations of carbohydrates, lipids and/or proteins, suchas proteoglycans (protein cores with attached side chains of chondroitinsulfate, dermatan sulfate, heparin, heparan sulfate, and/or keratansulfate); glycoproteins [e.g., selectins, immunoglobulins, hormones suchas human chorionic gonadotropin, Alpha-fetoprotein and Erythropoietin(EPO), etc.]; proteolipids (e.g., N-myristoylated, palmitoylated andprenylated proteins); and glycolipids (e.g., glycoglycerolipids,glycosphingolipids, glycophosphatidylinositols, etc.); (f) biologicallyderived homopolymers, such as polylactic and polyglycolic acids andpoly-L-lysine; (g) nucleic acids (e.g., DNA, RNA, etc.); (h) hormones(e.g., anabolic steroids, sex hormones, insulin, angiotensin, etc.); (i)enzymes (types: oxidoreductases, transferases, hydrolases, lyases,isomerases, ligases; examples: trypsin, collegenases, matrixmetallproteinases, etc.); (j) pharmaceuticals (e.g., beta blockers,vasodilators, vasoconstrictors, pain relievers, gene therapy, viralvectors, anti-inflammatories, etc.); (k) cell surface ligands andreceptors (e.g., integrins, selectins, cadherins, etc.); (l)cytoskeletal filaments and/or motor proteins (e.g., intermediatefilaments, microtubules, actin filaments, dynein, kinesin, myosin,etc.), or any combination thereof. For example, a biopolymer can beselected from the group consisting of fibronectin, vitronectin, laminin,collagen, fibrinogen, silk or silk fibroin.

In some embodiments of the compositions and methods described herein,cells engineered to express or secrete a multimodal TRAIL agent areencapsulated in an extracellular matrix comprising a thiol-modifiedhyaluronic acid and a thiol-reactive cross-linker, such as, for example,polyethylene glycol diacrylate.

In some embodiments of the compositions and methods described herein,cells engineered to express or secrete a multimodal TRAIL agent areencapsulated within permeable membranes prior to implantation. Severalmethods of cell encapsulation can be employed. In some embodiments,cells will be individually encapsulated. In other instances, many cellswill be encapsulated within the same membrane. Several methods of cellencapsulation are well known in the art, such as described in EuropeanPatent Publication No. 301,777, or U.S. Pat. Nos. 4,353,888, 4,744,933,4,749,620, 4,814,274, 5,084,350, and 5,089,272.

In one method of cell encapsulation, the isolated cells are mixed withsodium alginate and extruded into calcium chloride so as to form gelbeads or droplets. The gel beads are incubated with a high molecularweight (e.g., MW 60-500 kDa) concentration (0.03-0.1% w/v) polyaminoacid (e.g., poly-L-lysine) to form a membrane. The interior of theformed capsule is re-liquified using sodium citrate. This creates asingle membrane around the cells that is highly permeable to relativelylarge molecules (MW .about.200-400 kDa), but retains the cells inside.The capsules are incubated in physiologically compatible carrier forseveral hours in order that the entrapped sodium alginate diffuses outand the capsules expand to an equilibrium state. The resultingalginate-depleted capsules is reacted with a low molecular weightpolyamino acid which reduces the membrane permeability (MW cut-off 40-80kDa).

Other exemplary materials suitable for use in matrices and scaffoldsinclude, but are not limited to, PEG diacylate, hyaluronic acid,polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid(PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide(PLGA), polydioxanone, polygluconate, polylactic acid-polyethylene oxidecopolymers, modified cellulose, collagen, polyhydroxybutyrate,polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid),polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyaminoacids, polyorthoesters, polyacetals, polycyanoacrylates, degradableurethanes, aliphatic polyester polyacrylates, polymethacrylate, acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinylimidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinylalcohol, Teflon, nylon silicon, and shape memory materials, such aspoly(styrene-block-butadiene), polynorbornene, hydrogels, metallicalloys, and oligo(-caprolactone)diol as switchingsegment/oligo(p-dioxyanone)diol as physical crosslink. Other suitablepolymers can be obtained by reference to The Polymer Handbook, 3rdedition (Wiley, N.Y., 1989), the contents of which are hereinincorporated in their reference by entirety.

In some embodiments, additional bioactive substances can be added to abiopolymer matrix or scaffold comprising the cells engineered to expressa multimodal TRAIL agent described herein, such as, but not limited to,demineralized bone powder as described in U.S. Pat. No. 5,073,373 thecontents of which are incorporated herein by reference; collagen,insoluble collagen derivatives, etc., and soluble solids and/or liquidsdissolved therein; antiviricides, particularly those effective againstHIV and hepatitis; antimicrobials and/or antibiotics such aserythromycin, bacitracin, neomycin, penicillin, polymycin B,tetracyclines, biomycin, chloromycetin, and streptomycins, cefazolin,ampicillin, azactam, tobramycin, clindamycin and gentamycin, etc.;biocidal/biostatic sugars such as dextran, glucose, etc.; amino acids;peptides; vitamins; inorganic elements; co-factors for proteinsynthesis; hormones; endocrine tissue or tissue fragments; synthesizers;enzymes such as alkaline phosphatase, collagenase, peptidases, oxidases,etc.; polymer cell scaffolds with parenchymal cells; angiogenic agentsand polymeric carriers containing such agents; collagen lattices;antigenic agents; cytoskeletal agents; cartilage fragments; living cellssuch as chondrocytes, bone marrow cells, mesenchymal stem cells; naturalextracts; genetically engineered living cells or otherwise modifiedliving cells; expanded or cultured cells; DNA delivered by plasmid,viral vectors or other means; tissue transplants; demineralized bonepowder; autogenous tissues such as blood, serum, soft tissue, bonemarrow, etc.; bioadhesives; bone morphogenic proteins (BMPs);osteoinductive factor (IFO); fibronectin (FN); endothelial cell growthfactor (ECGF); vascular endothelial growth factor (VEGF); cementumattachment extracts (CAE); ketanserin; human growth hormone (HGH);animal growth hormones; epidermal growth factor (EGF); interleukins,e.g., interleukin-1 (IL-1), interleukin-2 (IL-2); human alpha thrombin;transforming growth factor (TGF-beta); insulin-like growth factors(IGF-1, IGF-2); platelet derived growth factors (PDGF); fibroblastgrowth factors (FGF, BFGF, etc.); periodontal ligament chemotacticfactor (PDLGF); enamel matrix proteins; growth and differentiationfactors (GDF); hedgehog family of proteins; protein receptor molecules;small peptides derived from growth factors above; bone promoters;cytokines; somatotropin; bone digestors; antitumor agents; cellularattractants and attachment agents; immuno-suppressants; permeationenhancers, e.g., fatty acid esters such as laureate, myristate andstearate monoesters of polyethylene glycol, enamine derivatives,alpha-keto aldehydes, etc.; and nucleic acids. The amounts of suchoptionally added bioactive substances can vary widely with optimumlevels being readily determined in a specific case by routineexperimentation.

Cancer

The limited availability of non-invasive methods to monitor multiplemolecular events has been one of the main limitations in testing theefficacy of various therapy paradigms against malignant conditions. Themultimodal TRAIL agents, and cells engineered to express these agents,described herein permit simultaneous therapeutic and diagnosticfunctionalities for use in developing and monitoring cancer therapies.Accordingly, provided herein are methods to treat a subject having amalignant condition comprising administering an effective amount of apharmaceutical composition comprising a multimodal TRAIL agent, or cellsengineered to express or secrete a multimodal TRAIL agent. In someembodiments of these methods, the cells engineered to express or secretea multimodal TRAIL agent are stem cells. In some such embodiments, thecells are neural stem cells. In some embodiments, the cells engineeredto express or secrete a multimodal TRAIL agent are encapsulated in amatrix.

The terms “malignancy,” “malignant condition,” “cancer,” or “tumor,” asused herein, refer to an uncontrolled growth of cells which interfereswith the normal functioning of the bodily organs and systems. A subjectthat has a malignancy (i.e., cancer or a tumor) is a subject havingobjectively measurable malignant or cancer cells present in thesubject's body. Included in this definition are benign and malignantcancers, as well as dormant tumors or micrometastatses. Cancers whichmigrate from their original location and seed vital organs caneventually lead to the death of the subject through the functionaldeterioration of the affected organs. Hemopoietic cancers, such asleukemia, are able to out-compete the normal hemopoietic compartments ina subject, thereby leading to hemopoietic failure (in the form ofanemia, thrombocytopenia and neutropenia) ultimately causing death.

By “metastasis” is meant the spread of cancer from its primary site toother places in the body. Cancer cells can break away from a primarytumor, penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. Metastasis can be local or distant. Metastasis isa sequential process, contingent on tumor cells breaking off from theprimary tumor, traveling through the bloodstream, and stopping at adistant site. At the new site, the cells establish a blood supply andcan grow to form a life-threatening mass. Both stimulatory andinhibitory molecular pathways within the tumor cell regulate thisbehavior, and interactions between the tumor cell and host cells in thedistant site are also significant.

Metastases are most often detected through the sole or combined use ofmagnetic resonance imaging (MRI) scans, computed tomography (CT) scans,blood and platelet counts, liver function studies, chest X-rays and bonescans in addition to the monitoring of specific symptoms.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include, but are not limited to, basal cell carcinoma, biliarytract cancer; bladder cancer; bone cancer; brain and CNS cancer; breastcancer; cancer of the peritoneum; cervical cancer; choriocarcinoma;colon and rectum cancer; connective tissue cancer; cancer of thedigestive system; endometrial cancer; esophageal cancer; eye cancer;cancer of the head and neck; gastric cancer (including gastrointestinalcancer); glioblastoma (GBM); hepatic carcinoma; hepatoma;intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung); lymphoma including Hodgkin's and non-Hodgkin'slymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g.,lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer;prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancerof the respiratory system; salivary gland carcinoma; sarcoma; skincancer; squamous cell cancer; stomach cancer; testicular cancer; thyroidcancer; uterine or endometrial cancer; cancer of the urinary system;vulval cancer; as well as other carcinomas and sarcomas; as well asB-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma(NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

In some embodiments of the methods described herein, a subject having amalignant condition has a brain cancer, brain tumor, or intracranialneoplasm. Intracranial neoplasms or cancers can arise from any of thestructures or cell types present in the CNS, including the brain,meninges, pituitary gland, skull, and even residual embryonic tissue.The overall annual incidence of primary brain tumors in the UnitedStates is 14 cases per 100,000. The most common primary brain tumors aremeningiomas, representing 27% of all primary brain tumors, andglioblastomas, representing 23% of all primary brain tumors (whereasglioblastomas account for 40% of malignant brain tumor in adults). Manyof these tumors are aggressive and of high grade. Primary brain tumorsare the most common solid tumors in children and the second mostfrequent cause of cancer death after leukemia in children.

In some embodiments of the methods described herein, a subject having amalignant condition has a glioma or glioblastoma (GBM). Gliomas arebrain tumors originating from glial cells in the nervous system. “Glialcells,” commonly called neuroglia or simply glia, are non-neuronal cellsthat provide support and nutrition, maintain homeostasis, form myelin,and participate in signal transmission in the nervous system. The twomost important subgroups of gliomas are astrocytomas andoligodendrogliomas. Belonging to the subgroup of astrocytomas,glioblastoma multiforme (referred to as glioblastoma hereinafter) is themost common malignant brain tumor in adults and accounts forapproximately 40% of all malignant brain tumors and approximately 50% ofgliomas. It aggressively invades the central nervous system and isranked at the highest malignancy level (grade IV) among all gliomas.Although there has been steady progress in their treatment due toimprovements in neuroimaging, microsurgery, diverse treatment options,such as temozolomide or radiation, glioblastomas remain incurable. Thelethal rate of this brain tumor is very high: the average lifeexpectancy is 9 to 12 months after first diagnosis. The 5-year survivalrate during the observation period from 1986 to 1990 was 8.0%. To date,the five-year survival rate following aggressive therapy, includinggross tumor resection, is still less than 10%.

Glioblastoma is the most common primary brain tumor in adults with avery poor prognosis. Treatment for GBM is maximal surgical tumorresectionor “debulking” followed by radiation therapy, with concomitantand adjuvant chemotherapy. However, recurrence rates of GBM and theassociated patient mortality are nearly 100%. Although resection of theprimary tumor mass has shown clinical benefit, adjuvant chemotherapy hasprovided limited extra benefit. One of the main impediments to theefficient delivery of many therapeutic molecules is the blood brainbarrier and vascular dysfunction in the tumor, which prevent many drugsfrom reaching brain tumor cells. Additionally, many drugs have shortsystemic half-lives and peak concentrations, which prevent drugs fromultimately reaching the brain and accumulating to therapeuticconcentrations in individual brain tumor cells. Tumor cells ofglioblastomas are the most undifferentiated ones among brain tumors, sothe tumor cells have high potential of migration and proliferation andare highly invasive, leading to very poor prognosis. Glioblastomas leadto death due to rapid, aggressive, and infiltrative growth in the brain.The infiltrative growth pattern is responsible for the unresectablenature of these tumors. Glioblastomas are also relatively resistant toradiation and chemotherapy, and, therefore, post-treatment recurrencerates are high. In addition, the immune response to the neoplastic cellsis rather ineffective in completely eradicating all neoplastic cellsfollowing resection and radiation therapy.

Glioblastoma is classified into primary glioblastoma (de novo) andsecondary glioblastoma, depending on differences in the gene mechanismduring malignant transformation of undifferentiated astrocytes or glialprecursor cells. Secondary glioblastoma occurs in a younger populationof up to 45 years of age. During 4 to 5 years, on average, secondaryglioblastoma develops from lower-grade astrocytoma throughundifferentiated astrocytoma. In contrast, primary glioblastomapredominantly occurs in an older population with a mean age of 55 years.Generally, primary glioblastoma occurs as fulminant glioblastomacharacterized by tumor progression within 3 months from the start withno clinical or pathological abnormalities.

Glioblastoma migrates along myelinated nerves and spreads widely in thecentral nervous system. In most cases surgical treatment shows onlylimited sustainable therapeutic effect. Malignant glioma cells evadedetection by the host's immune system by producing immunosuppressiveagents that impair T cell proliferation and production of theimmune-stimulating cytokine IL-2.

Accordingly, in some embodiments of the methods described herein, asubject having a malignant condition has or has had a glioblastoma. Insome such embodiments, the composition comprising a multimodal TRAILagent or cell engineered to express a multimodal TRAIL agentisadministered to the subject during or following a surgical procedure,such as a gross tumor resection. In some such embodiments, thecomposition comprising a multimodal TRAIL agent or cell engineered toexpress a multimodal TRAIL agentis is directly administered to a grosstumor resection site.

In some embodiments, the methods further comprise administering thepharmaceutical composition comprising a multimodal TRAIL agent, or cellsengineered to express or secrete a multimodal TRAIL agent, to a subjecthaving a malignant condition, such as a brain tumor (e.g.,glioblastoma), along with one or more additional chemotherapeuticagents, biologics, drugs, or treatments as part of a combinatorialtherapy. In some such embodiments, the chemotherapeutic agent biologic,drug, or treatment is selected from the group consisting of: radiationtherapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin,bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin,ABT-737, and PI-103. In some embodiments, the biologic can comprise asequence encoding a microRNA or RNA-based inhibitor molecule, such as aninhibitor RNA or iRNA.

In some embodiments of the methods described herein, the methods furthercomprise administering one or more chemotherapeutics agent to thesubject being administered the pharmaceutical composition comprising amultimodal TRAIL agent, or cells engineered to express or secrete amultimodal TRAIL agent. Non-limiting examples of chemotherapeutic agentscan include alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammalI and calicheamicinomegaIl (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above.

In addition, the methods of treatment can further include the use ofradiation or radiation therapy. Further, the methods of treatment canfurther include the use of surgical treatments.

As used herein, the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with, a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorderassociated with a cancer. Treatment is generally “effective” if one ormore symptoms or clinical markers are reduced. Alternatively, treatmentis “effective” if the progression of a disease is reduced or halted.That is, “treatment” includes not just the improvement of symptoms ormarkers, but also a cessation of at least slowing of progress orworsening of symptoms that would be expected in absence of treatment.Beneficial or desired clinical results include, but are not limited to,alleviation of one or more symptom(s), diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. The term “treatment” of a disease alsoincludes providing relief from the symptoms or side-effects of thedisease (including palliative treatment).

For example, in some embodiments, the methods described herein compriseadministering an effective amount of the multimodal TRAIL agents orcells engineered to express or secrete a multimodal TRAIL agentdescribed herein to a subject in order to alleviate a symptom of acancer. As used herein, “alleviating a symptom of a cancer” isameliorating any condition or symptom associated with the cancer. Ascompared with an equivalent untreated control, such reduction or degreeof prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or100% as measured by any standard technique.

The term “effective amount” as used herein refers to the amount of amultimodal TRAIL agent or cells engineered to express or secrete amultimodal TRAIL agent needed to alleviate at least one or more symptomof the disease or disorder, and relates to a sufficient amount ofpharmacological composition to provide the desired effect. The term“therapeutically effective amount” therefore refers to an amount of amultimodal TRAIL agent or cells engineered to express or secrete amultimodal TRAIL agent using the methods as disclosed herein, that issufficient to effect a particular effect when administered to a typicalsubject. An effective amount as used herein would also include an amountsufficient to delay the development of a symptom of the disease, alterthe course of a symptom disease (for example but not limited to, slowthe progression of a symptom of the disease), or reverse a symptom ofthe disease. Thus, it is not possible to specify the exact “effectiveamount”. However, for any given case, an appropriate “effective amount”can be determined by one of ordinary skill in the art using only routineexperimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD50/ED50. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC50 (i.e., theconcentration of the multimodal TRAIL agent), which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture, orin an appropriate animal model. Levels in plasma can be measured, forexample, by high performance liquid chromatography. The effects of anyparticular dosage can be monitored by a suitable bioassay. The dosagecan be determined by a physician and adjusted, as necessary, to suitobserved effects of the treatment.

Some aspects and embodiments disclosed herein can be illustrated by, forexample any of the following numbered paragraphs:

-   -   1. A multimodal TRAIL agent comprising a reporter module and a        therapeutic TRAIL module, wherein said therapeutic TRAIL module        comprises an extracellular domain of human TRAIL.    -   2. The multimodal TRAIL agent of paragraph 1, wherein the        extracellular domain of human TRAIL comprises amino acids        114-281 of SEQ ID NO: 1.    -   3. The multimodal TRAIL agent of any one of paragraphs 1 or 2,        further comprising a signal sequence.    -   4. The multimodal TRAIL agent of paragraph 3, wherein the signal        sequence comprises SEQ ID NO: 2.    -   5. The multimodal TRAIL agent of any one of paragraphs 1-4,        wherein the therapeutic TRAIL module further comprises an        isoleucine zipper domain.    -   6. The multimodal TRAIL agent of any one of paragraphs 1-5,        further comprising a linker domain C-terminal to the reporter        module and N-terminal to the therapeutic TRAIL module.    -   7. The multimodal TRAIL agent of paragraph 6, wherein the linker        domain comprises at least eight amino acids.    -   8. The multimodal TRAIL agent of paragraph 6, wherein the linker        domain comprises the amino acid sequence of SEQ ID NO: 4.    -   9. A pharmaceutical composition comprising the multimodal TRAIL        agent of any one of paragraphs 1-8 and a pharmaceutically        acceptable carrier.    -   10. A vector comprising a nucleic acid sequence encoding the        multimodal TRAIL agent of any one of paragraphs 1-8.    -   11. The vector of paragraph 10, wherein the vector is a        lentiviral vector or an adenoviral vector.    -   12. A cell comprising the nucleic acid sequence encoding the        multimodal TRAIL agent of any of paragraphs 1-8.    -   13. A cell comprising the vector of any one of paragraphs 10 or        11.    -   14. The cell of any one of paragraphs 12 or 13, wherein the cell        is a stem cell.    -   15. The cell of paragraph 14, wherein the stem cell is a neural        stem cell or a mesenchymal stem cell.    -   16. The cell of any one of paragraphs 12-15, wherein the cell is        encapsulated in a matrix or scaffold.    -   17. The cell of paragraph 16, wherein the matrix comprises a        synthetic extracellular matrix.    -   18. The cell of any one of paragraphs 16 or 17, wherein the        matrix is biodegradeable.    -   19. The cell of any one of paragraphs 17 or 18, wherein the        synthetic extracellular matrix comprises a thiol-modified        hyaluronic acid and a thiol reactive cross-linker molecule.    -   20. The cell of paragraph 19, wherein the thiol reactive        cross-linker molecule is polyethylene glycol diacrylate.    -   21. A composition comprising an isolated somatic cell that        comprises an exogenously introduced nucleic acid encoding a        multimodal TRAIL agent of any one of paragraphs 1-8 operably        linked to at least one regulatory sequence.    -   22. The composition of paragraph 21, wherein the isolated        somatic cell is an adult stem cell.    -   23. The composition of paragraph 22, wherein the adult stem cell        is a neural stem cell or a mesenchymal stem cell.    -   24. The composition of paragraph 23, wherein the neural stem        cell is generated from a pluripotent stem cell.    -   25. The composition of any one of paragraphs 21-24, wherein the        isolated somatic cell is encapsulated in a matrix or scaffold.    -   26. The composition of paragraph 25, wherein the matrix        comprises a synthetic extracellular matrix.    -   27. The composition of any one of paragraphs 25 or 26, wherein        the matrix is biodegradeable.    -   28. The composition of any one of paragraphs 26 or 27, wherein        the synthetic extracellular matrix comprises a thiol-modified        hyaluronic acid and a thiol reactive cross-linker molecule.    -   29. The composition of paragraph 29, wherein the thiol reactive        cross-linker molecule is polyethylene glycol diacrylate.    -   30. A method of treating a subject having a malignant condition        comprising administering a therapeutically effective amount of        the pharmaceutical composition of paragraph 9.    -   31. A method of treating a subject having a malignant condition        comprising administering a therapeutically effective amount of        the cells of any of paragraphs 12-20 or the composition of        paragraphs 21-29.    -   32. The method of any one of paragraphs 30 or 31, wherein the        malignant condition is a glioblastoma.    -   33. The method of any one of paragraphs 30-32, further        comprising administering to the subject, one or more additional        chemotherapeutic agents, biologics, drugs, or treatments as part        of a combinatorial therapy.    -   34. The method of paragraph 33, wherein the chemotherapeutic        agent, biologic, drug, or treatment is selected from the group        consisting of: radiation therapy, tumor resection surgery,        gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib,        AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737,        and PI-103.    -   35. The method of any one of paragraphs 30-34, wherein the        pharmaceutical composition of paragraph 9, the cells of any of        paragraphs 12-20, or the composition of paragraphs 21-29 is        administered at a surgical site.    -   36. The method of paragraph 35, wherein the surgical site is a        tumor resection site.    -   37. The pharmaceutical composition of paragraph 9 for use in a        method of treating a malignant condition.    -   38. The cells of any one of paragraphs 12-20 for use in a method        of treating a malignant condition.    -   39. The composition of any one of paragraphs 21-29 for use in a        method of treating a malignant condition.    -   40. The use of any of paragraphs 37-39, wherein the malignant        condition is a glioblastoma.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology, andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 18th Edition, published by Merck Research Laboratories, 2006(ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006. Definitions of common terms inmolecular biology are found in Benjamin Lewin, Genes IX, published byJones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew etal. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982);Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989);Davis et al., Basic Methods in Molecular Biology, Elsevier SciencePublishing, Inc., New York, USA (1986); or Methods in Enzymology: Guideto Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. KimmerlEds., Academic Press Inc., San Diego, USA (1987); Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et. al., ed., John Wiley and Sons, Inc.) and Current Protocolsin Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons,Inc.), which are all incorporated by reference herein in theirentireties.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that could beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

EXAMPLES Example 1: A MultiModal TRAIL Agent Integrating Therapeutic andDiagnostic Activities Reveals Multiple Aspects of Stem Cell-BasedTherapy Engineering and Screening of Optimized Therapeutic andDiagnostic Luciferase-S-TRAIL Fusions

To create novel multimodal TRAIL agents that can both be easilyvisualized for serial monitoring of cell-based pharmacokinetics andretain potent anti-tumor function, numerous lentiviral vectors (LV)encoding fusion molecules between S-TRAIL, i.e., a therapeutic,secretable TRAIL module, and various luciferases, as reporter modules,were generated. (FIG. 1, FIG. 6A-6D, Table 1). To select the moleculewith optimized diagnostic and therapeutic activity, 293T cells weretransduced with LV encoding each construct at multiplicity of infection(MOI) 1 (FIG. 6B), and conditioned media from transfected cells wasscreened for light emission, S-TRAIL concentration, and anti-tumorefficacy. To first select the appropriate molecular composition andorientation, direct fusions of firefly luciferase (FLuc), Renillaluciferase (RLuc), or Gaussia princeps luciferase (GpLuc) to theC-terminus of S-TRAIL were screened (FIG. 1, constructs 1-3). Of thesefusions, only TRAIL-GpLuc (TRGp, construct 3) showed light emission bybioluminescence imaging on media from transduced cells; however, noS-TRAIL was detected in the conditioned media and no killing ofGli36-EGFRvIII human cancer cells was observed from TRGp-containingmedia (FIG. 1, construct 3; Table 1). In contrast, when GpLuc was fusedto the N-terminus of S-TRAIL (GpTR, construct 4), detectable levels oflight emission, S-TRAIL concentration, and reduction in tumor cellviability were observed (FIG. 1, Table 1).

As GpTR showed maximal diagnostic and therapeutic activity, the effectsof intramolecular spacing were investigated by engineering GpLuc andS-TRAIL fusions separated by two different linker modules. As shown inFIG. 1, increased intramolecular spacing increased the function of bothGpLuc and S-TRAIL as the fusion variant containing linker-2 (GpL2TR,construct 6) showed markedly increased diagnostic and therapeuticactivity compared to the GpTR direct fusion.

Lastly, to maximize delivery by optimizing extracellular secretion, twoadditional multimodal TRAIL fusion variants were engineered in which theendogenous secretion sequence was replaced by the signal sequence fromFlt3 ligand, a sequence known to induce highly efficient proteinsecretion (Shah et al. Cancer Research 2004) (FIG. 1). The first variantwas based on GpL2TR (SGpL2TR, construct 7). In the second (SRLucOL2TR,construct 8), GpLuc was replaced by an alternative form of Renillaluciferase (RlucO) optimized for extracellular light production andsuggested to have increased in vivo luciferase activity compared toGpLuc (Venisnik et al. Mol Imaging Biol 2007). The results showed thatthe modified secretion sequence in SGpL2TR led to over a 1.5-foldincrease in photon emission compared to that in GpL2TR (FIG. 1, Table1). Similarly, greater S-TRAIL concentration and tumor cell killing wereobserved by SGpL2TR. Although SRLucOL2TR demonstrated significantdiagnostic and therapeutic activity, in vitro levels of bioluminescencesignal, S-TRAIL concentration, and tumor cell killing were less thanthose observed for SGpL2TR yet greater than or equal to those for GpL2TR(FIG. 1, Table 1).

Characterizing Optimized Luciferase-S-TRAIL Fusions In Vivo

As the results above indicate, the multimodal TRAIL agents SGpL2TR andSRLucOL2TR described herein had the greatest diagnostic and therapeuticactivities in vitro, so they were investigated for their activity invivo. To first confirm proper expression of the proteins, Western blotanalysis was performed on lysates from transduced cells using ananti-TRAIL antibody. As shown in FIG. 2A, both SGpL2TR and SRLucOL2TRwere detected at high levels and at the anticipated size. For in vivocharacterization of SGpL2TR and SRLucOL2TR, U251 cancer cells that havebeen previously shown to have a slow rate of TRAIL-induced cell death(Kock et al. Neoplasia 2007, Hingtgen et al. Mol Cancer Ther 2008) wereutilized. To track changes in tumor volume, U251 cells were firsttransduced with LV vectors encoding GFP-FLuc, followed by transductionwith SGpL2TR, SRLOL2TR, or control virus (FIG. 2B, FIG. 6A-6D).Following subcutaneous implantation, GpLuc or RLucO imaging wasperformed to track fusion proteins and Fluc imaging was performed tomonitor changes in tumor volume. As shown in FIGS. 2C-2D, SGpL2TR orSRLucOL2TR imaging on days 1, 3, and 15 demonstrated stable secretion ofthe luciferase-S-TRAIL fusion proteins from the infected cells whenexpressed relative to FLuc photon emission (FIG. 2C). This stabledelivery coincided with a gradual yet significant reduction in tumorvolume determined by FLuc imaging on days 2, 7, and 15 (FIG. 2D).Interestingly, in contrast to the results observed in vitro, tumorstransduced with SRLOL2TR showed greater photon emission thanSGpL2TR-transduced tumor in vivo. The enhanced photon emission byRLucO-containing fusion was not due to differences in TRAIL-inducedreduction in tumor volume, as further side-by-side in vivo comparisonusing nontherapeutic forms of SGpLuc and SRLucO confirmed greater lightemission from RLucO tumors (FIG. 2E, FIGS. 7A-7D). As both SGpL2TR andSRLOL2TR display similar anti-tumor activity, yet SRLOL2TR showsenhanced light emission allowing improved in vivo detection (datasummarized in Table 1), all further studies described in this Exampleutilized SRLOL2TR. Together, these results demonstrate the engineeringand optimization of SRLOL2TR to follow delivery and therapeutic efficacyof the multimodal TRAIL agent protein in vitro and in vivo.Additionally, they demonstrate the importance of molecular organization,intermolecular spacing, optimized secretion, and in vivo activity indeveloping multifunctional and multimodal TRAIL molecules.

SRLOL2TR Reveals Stem Cell Lines Exhibit Different Secretion Kineticsthat Effect Cancer Cell Killing

Different stem cell lines were investigated for evidence of differentsecretion levels and duration of delivery that influence anti-tumorefficacy. To this end, three different stem cell lines, (1) primarymouse neural stem cells (mNSC), (2) human neural stem cells (hNSC), and(3) primary mouse mesenchymal stem cells (mMSC), were transduced withincreasing MOI of lentivirus (LV) encoding SRLOL2TR (FIGS. 3A-3B). Fortransduction of mNSC, cells were first seeded on coated tissue cultureplates to establish a monolayer that ensured efficient LV-mediatedtransduction (FIG. 3A). As shown in FIG. 3B, hNSC and mNSC were robustlytransduced at low MOI, whereas mMSC required MOI of 8 to reach the samepercentage of transduction as hNSC or mNSC. Serial bioluminescenceimaging performed on media from equally transduced cells showed that,despite early robust secretion of SRLOL2TR by hNSC (FIG. 8A), levelsrapidly declined and were nearly absent by 48 hours (FIG. 3C) due toTRAIL-induced death of hNSC. In contrast, mNSC and mMSC retained stablesecretion of SRLOL2TR through 14 days, although mNSC secretion ofSRLOL2TR was markedly greater than levels detected from mMSC (FIG. 8A).

To determine if differences in secretion between the three stem celllines translated to differences in anti-tumor cell efficacy, co-culturestudies were performed using different ratios of stem cells expressingSRLOL2TR and glioma lines with different sensitivities to TRAIL-mediatedapoptosis (Gli36-EGFRvIII, highly TRAIL sensitive; U251, less TRAILsensitive), both engineered to express mCherryFLuc. The imaging of hNSCsecretion showed extremely low levels of SRLOL2TR in the media at thetime point assayed, and this translated to minimal effects on eitherGli36-EGFRvII (FIG. 3D) or U251 (FIG. 3E) tumor cell viability. Imagingof media from mNSC showed robust levels of SRLOL2TR that increased asthe stem cell/tumor cell ratio increased, with a ratio of 0.04/1 leadingto detectable amounts of SRLOL2TR. Importantly, the efficient secretionof SRLOL2TR lead to stem cell/tumor cell ratios as low as 0.1/1 and0.2/1, significantly decreasing the viability of Gli36-EGFRvII (FIG. 3D)or U251 (FIG. 3E) cells, respectively, in a dose-dependent manner.Imaging of the third stem cell line, mMSC, showed markedly lower levelsof SRLOL2TR compared to mNSC, suggesting significantly lower delivery oftherapeutic proteins (FIGS. 3D-3E). Similarly, Gli36-EGFRvIII cellviability was decreased but required 10-fold more therapeutic mMSC toinduce killing compared to mNSCs (FIG. 3D), whereas U251 cells requireda mMSC/tumor ratio of 3/1 before a significant difference in tumor cellviability was detected (FIG. 3E). Together, these results show thatimaging of the multimodal TRAIL agent SRLOL2TR revealed markeddifferences in the delivery of therapeutics by hNSC, mNSC, and mMSC,which resulted in significant differences in the ability of each line toinduce tumor cell killing in human cancer cell lines both highlysensitive and less sensitive to TRAIL. Additionally, mNSC secreted thehighest levels of SRLOL2TR and induced the largest decrease in cancercell viability at the lowest stem cell/cancer cell ratio.

On the basis of these results, mNSC secreting SRLOL2TR were selected asthe stem cell line for further investigation. Western blot analysisconfirmed that mNSC did not express TRAIL death receptors (FIG. 3F).Immunocytochemistry revealed that these cells robustly expressed the NSCmarker Nestin (FIG. 3Ga) and possessed the capacity to generate cells ofneural lineage (FIGS. 3Gb-3Gc). In addition, when transduced stem cellswere implanted 1 mM adjacent to established Gli36-EGFRvIII human gliomasexpressing FLuc-DsRed2 (Gli36-EGFRvIII-FD; FIG. 8B) in the frontal lobeof mice, two distinct populations of green stem cells and red gliomaswere observed 2 days post-implantation (FIG. 3 Ha). However, stem cellswere observed specifically migrating towards the glioma by day 5(FIG. 3Hb) and showed accumulation in the tumor by day 10 (FIG. 3 Hc).Demonstrating the role of the extrinsic apoptotic cascade in stemcell-secreted SRLOL2TR-induced cancer cell death, Western blot analysisand luciferase-based assay on Gli36-EGFRvIII cells treated with SRLOL2TRconditioned media revealed significant activation of caspase 3/7 (FIG.31) and increases in cleavage of caspase-8 and Poly(ADP-ribose)polymerase (PARP) (FIG. 3J). These results show that transduced mNSCretain all the characteristics of NSC in vitro and also migratespecifically to gliomas in mice bearing intracranial tumors.

Stem Cell-Based Delivery Significantly Improves Pharmacokinetics andEfficacy of Therapies Determined by Noninvasive Imaging of SRLOL2TR

In clinics, numerous chemotherapies are administered to patients viai.v. infusion or direct injection, yet these methods can lead tosignificant levels of the drugs accumulating in normal organs, resultingin dose-limiting toxicities. In stem cell-based delivery, the cells aretypically engrafted around the tumor to provide sustained levels oftherapeutic protein for direct targeting of tumor cells. It wasdetermined if SRLOL2TR could be used to visualize differences betweenthe pharmacokinetics of delivery to tumors by mNSC and i.v. orintratumoral injection of purified protein in vivo in models designed tomimic the clinical settings. First, the bioluminescence signal from bothGli36-EGFRvIII-FD and mNSC engineered with SRLOL2TR were shown tocorrelate directly with cell number (FIGS. 9A-9B). Next, mice wereimplanted with mNSC expressing SRLOL2TR around establishedGli36-EGFRvIII-FD tumors to mimic clinical engraftment of cell-basedtherapies. RLucO imaging performed 24 hours after implantation of mNSCrevealed a robust bioluminescence signal that co-localized with theestablished tumor (FIG. 4A). FLuc bioluminescence imaging showed asignificant reduction in tumor volume by 48 hours (FIG. 4B). Ex vivoanalysis demonstrated RLucO signal was present in the excised tumor butabsent from other organs and tissues (FIG. 4C). Alternatively, whenmedia containing SRLOL2TR was delivered by i.v. injection to mimic thesystemic delivery of chemotherapies, a signal was detectable thatpersisted through 40 minutes but was absent by 24 hours (FIG. 4D).Furthermore, the bioluminescence signal was detected in the liver, lung,kidney, and blood as well as the tumor (FIG. 4E) and did not have anyeffect on tumor volume (FIG. 4F). Lastly, to determine if SRLOL2TR couldbe used to visualize differences in delivery of purified proteinadministered in the same manner as stem cells, media containing SRLOL2TRwas injected directly into established tumors. As shown in FIGS. 4G-41,the direct injection of media resulted initially in a robust signalpresent at the site injection that gradually decreased to baseline nearbaseline levels by 40 minutes and was entirely absent at 24 hours (FIG.4G). Similar to i.v. infusion, the direct injection of media to thetumor led to detectable accumulation of the SRLOL2TR in the liver, lung,and kidney in addition to the tumor (FIG. 4H) and also had minimaleffect on tumor volume (FIG. 4I).

The results described herein indicate that combining the novel SRLOL2TRprotein and optical imaging permits elucidation of differences inpharmacokinetics, tissues distribution, and therapeutic efficacy ofanti-cancer proteins delivered to tumors by engineered stem cells ori.v. injection. As SRLOL2TR allows visualization of therapeutic levelsin real-time, we show that a single administration of engineered stemcells provides continuous sustained and localized delivery oftherapeutics that attenuates tumor growth, whereas a single i.v.infusion or direct administration of media containing SRLOL2TR resultsin widespread off-target binding and significantly shortened deliverywindow that correlates with minimal anti-tumor effects.

Stem Cells Lead to Sustained Delivery of SRLOL2TR for Treatment ofHighly Malignant Intracranial Glioblastoma

Lastly, the above stem cell-based approaches were tested in anintracranial glioma model, a disease where effective delivery oftherapeutic agents is further limited by the blood-brain barrier. Tofirst investigate the survival of mNSC in the context of glioma, mNSCexpressing GFP-FLuc (FIG. 9C) were mixed with Gli36-EGFRvIII humanglioma cells and implanted intracranially in mice. FLuc bioluminescenceimaging revealed the presence of transduced mNSC in the brain, and thelevels remained constant through 15 days (FIG. 5A). After confirmingintracranial survival of transduced mNSC, therapeutic mNSC engineered toexpress SRLOL2TR were implanted intracranially in severe combinedimmunodeficiency (SCID) mice together with Gli36-EGFRvIII-FD. RLucOimaging on days 2, 6, 9, and 12 showed robust and stable delivery ofSRLOL2TR by mNSC (FIG. 5B). Serial FLuc imaging revealed that mNSCdelivery of SRLOL2TR led to marked attenuation in glioma progression,with significant decrease in FLuc signal in SRLOL2TR-treated mice by day6 (FIG. 5C). Post-mortem immunohistochemical analysis performed 4 dayspost-implantation confirmed the presence of GFP-expressing mNSC (FIG.5D) and demonstrated expression of the NSC marker Nestin (FIGS. 5Da,5De, 5Di). Furthermore, mNSC did not stain positive for the astrocytemarker glial fibrillary acidic protein (GFAP) (FIGS. 5Db, 5Df, 5Dj), theneuronal marker Tuj-1 (FIGS. 5Dc, 5Dg, 5Dk), or the proliferation markerKi67 (FIGS. 5D-d,h,l), which showed strong staining of the highlyproliferating glioma cells. By simultaneously monitoring therapeuticdelivery by mNSC in the brain and intracranial glioma volumes, theseresults show that therapeutic stem cells secreting SRLOL2TR areeffective anti-glioma therapies.

CONCLUSIONS

Demonstrated herein is the development of novel multifunctional,multimodal TRAIL agents as molecules that have both diagnostic (in vivotracking via optical reporters) and therapeutic (anti-tumor via thecytotoxic agent TRAIL) properties. Further, their application incharacterizing therapeutic delivery by engineered stem cells isdemonstrated. To develop molecules with the greatest optical reporteractivity and highest tumor cell toxicity, it is critical to select boththe appropriate luciferase and orientation for the fusion to TRAIL (N-or C-terminus). After screening fusion proteins containing severaldifferent luciferase proteins, the results showed fusion proteinscontaining GpLuc produced the greatest light emission in vitro, whileSRLucO containing fusions performed better in vivo.

Furthermore, it was shown that fusion of luciferase proteins to theN-terminus of TRAIL permitted retention of both the imaging propertiesof the luciferase and anti-tumor properties of TRAIL, whereas C-terminalfusions inactivated the multimodal TRAIL agent. Without wishing to bebound or limited by theory, the inactivation of S-TRAIL is most likelydue to the fact that the C-terminus of S-TRAIL contains the cell-bindingdomain of TRAIL (Wiley et al, Immunity 1995), and therefore, the fusionof proteins to the C-terminus of TRAIL either prevents proper folding ofthe protein or interferes with interaction of S-TRAIL with itsreceptors, DR4 and DRS, which is in agreement with the structure ofother TRAIL fusion proteins (Shen et al. Appl Microbiol Biotechnol 2007,Bremer et al. Neoplasia 2004). Furthermore, previous reports haveemphasized the importance of protein linkers in order to achieve optimalactivity of luciferase fusion proteins (Venisnik et al. Protein Eng DesSel 2006, Ray et al. Cancer Res 2003). In agreement with these reports,greater extracellular BLI signal, TRAIL levels, and cell killing wereobserved with the inclusion of longer intracellular linkers. Withoutwishing to be bound or limited by theory, the increased intramolecularspacing between the therapeutic, secretable module, S-TRAIL, and thereporter module, luciferase, by inclusion of linker-2 better preservedthe functionality of both the luciferase and S-TRAIL modules, thusleading to the observed increases in photon emission, S-TRAILconcentration, and cell killing. Taken together, the multimodal TRAILagent SRLOL2TR combines the potent anti-tumor properties of S-TRAIL withthe simple noninvasive assessment of therapeutic delivery afforded byluciferase imaging.

One of the primary challenges to achieving effective anti-tumor therapyis highly efficient delivery of the anti-tumor agent specifically to thetumor, while minimizing toxicity to nonmalignant tissue. Although simpleto administer, systemic administration of therapies often leads toaccumulation of the toxic compounds at high levels in the liver andkidneys, resulting in dose-limiting renal- and hepatotoxicity (Kelley etal. J Pharmacol Exp Ther 2001, Lin, Drug Metab Dispos 1998). TRAIL hasbeen shown to have minimal cytotoxic effects on normal tissue; however,its short half-life and accumulation after systemic injection have beenlimitations to its potential use in clinics (Ashkenazi et al., J ClinOncol 2008). Because of their potential to migrate to sites of diseaseand integrate into the cytoarchitecture of the brain, stem cells (NSC,MSC) have received much interest for the treatment of numerousneurologic disorders (Corsten and Shah, Lancet Oncology 2008, Singec etal. Annu Rev Med 2007). Previous studies from our lab and othersdemonstrated that NSC and human MSC migrate extensively throughout themurine brain and exhibit an inherent capacity to home to establishedgliomas (Sasportas et al. Proc Natl Acad Sci 2009, Shah et al Ann Neurol2005, Shah et al. J Neurosci 2008). Stem cells armed with S-TRAILinhibited progression of gliomas in a xenogenic transplant model(Sasportas et al. Proc Natl Acad Sci 2009, Shah et al Ann Neurol 2005);however, assessing the pharmacokinetics of the molecules released bytherapeutic NSC has been difficult.

As described herein, the combination of SRLOL2TR and real-time imagingdemonstrated the advantages of stem cell-based delivery. Noninvasivemonitoring of SRLOL2TR pharmacokinetics revealed a markedly increaseddelivery time and reduced nonspecific biodistribution that culminated ineffective reduction in tumor burden. In contrast, i.v. or intratumoralinjection of SRLOL2TR resulted in rapid clearance, widespreadbiodistribution, and minimal effects on the tumor. The results providedherein permit the first real-time comparison of pharmacokinetics whentherapies are delivered by stem cells or systemic administration.

The ability to serially monitor the level of therapeutic proteindelivered by stem cells, such as NSCs, is critical to effectivecell-based therapy. Longitudinal imaging of therapies permitsconfirmation of the initial levels delivered, permits confirmation ofwhether there is a need to increase dose by injection of additionaltherapeutic NSC should inhibition of tumor growth not be observed, orindicates the need for re-administration if the dose begins to decline.The examples shown herein of intracranial glioblastoma xenograft usingthe combination of mNSC and SRLOL2TR revealed robust and sustaineddelivery of TRAIL fusion as early as 2 days post-implantation and showedthat sustained SRLOL2TR persisted through day 12. Importantly, thecontinuous delivery of SRLOL2TR by stem cells markedly decreased gliomaburden as early as day 6. These results demonstrate the effectiveness ofstem cell-mediated delivery of SRLOL2TR as an anti-glioma therapy, assignificant tumor regression was achieved with a single administrationof therapeutically engineered stem cells. In the event that SRLOL2TRlevels decrease at time points beyond those described herein, thediagnostic functionality of SRLOL2TR would reveal these changes, andstem cells could be re-administered to ensure continued suppression oftumor growth.

In conclusion, shown herein is the engineering of novel fusionmultimodal TRAIL agents with both diagnostic and therapeuticfunctionalities, and utilization of the novel multimodal TRAIL agentSRLOL2TR as a means to determine vital aspects of stem cell-basedtherapies. These studies showed, in part, how differences in deliveryefficiency between different cell lines affected their therapeuticapplication, how improved pharmacokinetics mediated by stem celldelivery influenced anti-tumor efficacy of a therapy, and how selectionof the optimal therapeutic stem cell line can effectively attenuatehighly malignant tumors in vivo.

Example 2: Encapsulated Therapeutic Stem Cells Transplanted in the TumorResection Cavity Eradicate Brain Tumors Creating a Mouse Model of GBMResection

To develop a mouse surgical resection model of GBM, malignant GBM cellsengineered with fluorescent and bioluminescent proteins were employed.Human GBM cells, U87 were transduced with lentiviral constructLV-Fluc-mCherry, sorted and screened for mCherry (FIG. 10A) and Flucexpression (FIG. 10B). A direct correlation was seen between the Flucexpression and the cell number (FIG. 10B). U87-Fluc-mCherry human GBMcells were implanted in a cranial window created by removal of a smallcircular portion of the skull (FIGS. 10C-10D, FIGS. 14A-14B) and micewere imaged for tumor progression/volumes over time by fluorescenceintravital microscopy (IVM) and Fluc bioluminescence imaging, (FIGS.10E, 10I). Established GBM tumors in mice generated by implantation oflow (7.5×10⁴) and high (1.5×10⁵) number of GBM cells were resected andIVM and BLI imaging post resection were used to determine the extent ofresection (FIGS. 10F, 10I). Fluc imaging confirmed greater than 60% ofthe tumor was resected in mice bearing small tumors, while over 80% ofthe tumor was resected in mice with large tumors that were easier tovisualize (FIG. 10I, FIG. 14C). High resolution IVM and IHC confirmedthe efficiency of resection (FIG. 10E-10I) and Kaplan-Meier survivalcurves showed a significant increase in the survival of resected tumormice as compared to the mice with un-resected tumors in both tumor types(FIG. 10J).

Characterizing Engineered mNSC Encapsulated in Biocompatible sECMs InVitro and In Vivo

To assess survival of NSC encapsulated in sECMs in vitro, mouse (m)NSCwere engineered to either express GFP-Fluc or to co-express GFP-Fluc anda secretable marker, Ss-Rluc(o) using our previously developeddiagnostic lentiviral vectors (Shah et al. J Neurosci 2008, Hingtgen etal. Mol Cancer Ther 2008) (FIG. 11A). We confirmed a direct correlationbetween different sECM encapsulated cell numbers and Fluc activity andRluc(o) activity in vitro (FIG. 15A). Both engineered mNSC types wereencapsulated in sECMs (FIG. 11B). A stable increase in both the cellproliferation (Fluc activity) and protein secretion (Rluc activity) wasseen when mNSC co-expressing GFP-Fluc and a secretable luciferase(Ss-Rluc(o)) encapsulated in sECM were cultured over-time (FIG. 11C). Toassess the influence of sECMs on cell survival in vivo, mNSC-GFP-Fluc insuspension or encapsulated in sECM were implanted intracranially andmice were imaged serially for mNSC survival by Fluc activity. Asignificant increase in cell viability was observed in mice bearing sECMencapsulated mNSC as compared to the non-encapsulated mNSC (FIG. 11D).To longitudinally monitor mNSC expressed proteins in vivo, weintracranially implanted sECM encapsulated mNSC co-expressing GFP-Flucand Ss-Rluc(o) in mice. In vivo, dual bioluminescence imaging showed astable production of proteins from mNSC over-time (FIG. 11E). In orderto follow migration of sECM encapsulated mNSC, mice bearingU87-Fluc-mCherry GBMs in a cranial window were implanted withmNSC-GFP-Rluc encapsulated in sECMs 1 mm away from an established tumor.Intravital imaging revealed that sECM encapsulated NSC migrate out ofthe sECMs and specifically home to tumors in the brain over a period of4 days (FIGS. 11F-11I).

To assess the therapeutic potential of mNSC expressing therapeuticproteins that specifically kill tumor cells, mNSC were engineered toexpress S-TRAIL, a cytotoxic agent that induces apoptosis specificallyin tumor cells, and its diagnostic variant Di-S-TRAIL or controls.S-TRAIL ELISA revealed high TRAIL concentration (150-650 ng/mL) in theculture medium containing mNSC-S-TRAIL cells encapsulated in sECMs (FIG.15B). A significant reduction in GBM cell viability was seen whenmNSC-S-TRAIL cells encapsulated in sECMs were placed in the culture dishcontaining human GBM cells U87-Fluc-mCherry (FIGS. 12A-12E). Thedecrease in GBM cell viability was associated with increase incaspase-3/7 activity (FIG. 12E) and changes in caspase-8 (FIG. 12F) andPARP activity (FIG. 12F, FIG. 19). S-TRAIL ELISA confirmed a high TRAILconcentration (150-650 ng ml-l) in the culture medium containingmNSC-S-TRAIL cells encapsulated in sECM (FIG. 15B). To simultaneouslymonitor release of S-TRAIL from sECM encapsulated mNSC and its effect onGBM cell viability in sECM encapsulated mNSC co-cultured withU87-mCherry-Fluc GBM cells, mNSC were engineered with our recentlycreated diagnostic variant of S-TRAIL (Di-S-TRAIL). Dual bioluminescenceimaging showed robust levels of Di-S-TRAIL released from sECM thatincreased as the stem cell/tumor cell ratio increased and resulted in asignificant and dose-dependent decrease in GBM cell viability (FIG.12G). These results demonstrate that sECM encapsulated engineered mNSCsurvive longer in mice brains, migrate to tumors in the brain and induceapoptosis in GBM cells.

Transplantation of ‘Armed’ mNSC into the Tumor Resection Cavity and InVivo Imaging of Tumor Regression

To assess survival of mNSC encapsulated in sECMs in vivo in mouse modelsof resection, mNSC-GFP-Fluc were implanted either in suspension orencapsulated in the resection cavity of U87 GBMs. sECM encapsulated mNSCwere retained in the tumor resection cavity (FIGS. 13A-13C) at highlocal concentrations adjacent to the residual tumor cells (FIG. 13D).sECM encapsulated mNSC survival in the tumor resection cavity over aperiod of 1 month was significantly higher as compared to thenon-encapsulated mNSC in the resection cavity (FIG. 13E). Next, toassess the therapeutic potential of sECM encapsulated mNSC-S-TRAIL inmouse resection models of GBM, sECM encapsulated mNSC-S-TRAIL ormNSC-GFP-Rluc were implanted intracranially in a resection cavity of themouse model of resection and mice were followed for changes in tumorvolume by serial Fluc bioluminescence imaging and for survival. As shownin FIG. 13F, sECM-encapsulated mNSC-S-TRAIL induced a dramatic increasein caspase-3/7 activity and greater than an 80% decrease in residualtumor cells as early as 3 days post-seeding that could be followed bysimultaneously visualizing caspase-3/7 activation and tumor volumes inreal time in vivo. Importantly, sECM-mNSC-S-TRAIL suppressed re-growthof residual tumor cells through 49 days post-resection (FIG. 16).Highlighting the survival benefit of this approach, mice treated withcontrol sECM-encapsulated mNSC-GFP-Rluc demonstrated a median survivalof 14.5 days GBM post-resection. In contrast, 100% of mice treated withmNSC-S-TRAIL encapsulated in sECM after GBM resection were alive 42 dayspost-treatment (FIG. 13G). sECM encapsulation was required for thesurvival benefit, as mNSC-S-TRAIL delivered in suspension into theresection cavity showed no significant increase in survival (FIG. 13G).These results demonstrate that sECM encapsulated therapeutic mNSC areretained in the tumor resection cavity, result in killing of residualGBM cells that significantly increase survival of mice.

Several studies have shown that freshly isolated primary glioma linesfrom clinical specimens more accurately recapitulate the clinicalscenario of GBMs. To assess the clinical relevance of sECM-encapsulatedstem cell-based therapeutic regimen in a more clinically relevant model,we used a TRAIL-sensitive primary human invasive glioma line, GBM8, andhuman bone marrow-derived MSCs (hMSCs). We engineered GBM8 cells toexpress a mCherry-Fluc fusion protein and showed that theGBM8-mCherry-Fluc line retained the tumor cell invasive properties ofthe parental line in culture (FIG. 17A) and in vivo (FIG. 17B). Therewas a direct correlation between the Fluc signal intensity and thenumber of cells in vitro in the ranges tested (FIG. 20). To assess themigration and the therapeutic potential of hMSCs expressing therapeuticproteins that specifically kill tumor cells, we engineered hMSCs toexpress GFP or S-TRAIL and GFP. In cultures of sECM-encapsulated hMSCswith GBM8 cells, hMSCs expressing GFP only or S-TRAIL migrated out ofthe sECM and tracked GBM8 cells (FIGS. 17C-17F). Furthermore,hMSC-S-TRAIL cells induced GBM8 cell death in a time-(FIG. 17G) andcaspase-3/7-dependent (2.7±0.1 fold increase in caspase-3/7 activity incomparison to hMSC-GFP cells) manner. Next, to assess the therapeuticpotential of sECM-encapsulated hMSC-S-TRAIL cells in mouse resectionmodels of primary GBM8 tumors, we implanted sECMencapsulatedhMSC-S-TRAIL or hMSC-GFP cells intracranially in a GBM8 tumor resectioncavity and followed mice for changes in tumor volume by serial Flucbioluminescence imaging. The presence of sECM-encapsulated hMSC-S-TRAILcells resulted in significantly less residual GBM8 cells than in thecontrols (FIG. 17H). Fluorescence imaging of brain sections revealed thepresence of encapsulated hMSCs in the tumor resection cavity and alsoindicated hMSCs migration to invading glioma cells (FIGS. 171-17J).Histopathological analysis on brain sections revealed a significantlyhigher number of cleaved caspase-3-positive cells inhMSC-S-TRAIL-treated mice than in controls (FIGS. 17K-17L; 4.2±0.2 foldincrease in the hMSC-S-TRAIL group versus the hMSC-GFP group). Theseresults show that sECM encapsulated engineered human MSCs havetherapeutic benefits against primary tumor-derived GBMs.

CONCLUSIONS

In this Example, diagnostic and therapeutic mNSCs encapsulated in sECMwere tested in a murine model of GBM resection. As demonstrated herein,sECM encapsulation of mNSC significantly increased retention time in theGBM resection cavity, permitted robust tumor-selective migration andallowed secretion of anti-tumor proteins from the sECM encapsulated stemcells in vivo. Mimicking the clinical scenario of GBM resection andsubsequent treatment, TRAIL-secreting sECM encapsulated mNSCtransplanted in the resection cavity eradicated residual tumor cells,delayed tumor re-growth and significantly increased survival of mice.Furthermore, we demonstrate herein that TRAIL-secretingsECM-encapsulated stem cells transplanted in the resection cavitysignificantly delayed tumor regrowth in mice bearing both established(U87) and primary invasive (GBM8) GBMs and significantly increasedsurvival of mice bearing established GBMs.

The clinical standard of care for patients suffering from glioblastomaincludes surgical debulking (Wen and Kesari, N Engl J Med 2008, Minnitiet al. J Neurooncol 2008, Bidros and Vogelbaum Neurotherapeutics 2009),yet nearly all pre-clinical models focus on treating established solidtumors. Previously, a limited number of studies have shown thefeasibility of resecting established GBMs in different animal models(Akbar et al. J Neurooncol 2009, de Oliveria et al. Neurooncol 2009). Inthis study, by integrating fluorescent and bioluminescent markers andextensive optical imaging, we simultaneously confirmed the presence ofestablished tumors, visualized the extent of tumor resection andserially monitored tumor re-growth post-resection. The inclusion ofpost-resection bioluminescence imaging permitted gross assessment oftotal tumor removal, while real-time fluorescent microscopy permittedvisualization of residual tumor cells and associated blood vessels inresected tumors. In recent years, in addition to the established GBMlines, human brain tumor tissue has been isolated and utilized forpre-clinical studies (Pandita et al. Genes Chromosomes Cancer 2004,Piccirillo et al. J Mol Med 2009, Wakimoto et al. Cancer Res 2009). Indeveloping this study, the use of both established and primary tumorlines that have previously extensively characterized for invasivenessand resistance/sensitivity to TRAIL mediated apoptosis was considered(Hingtgen et al. Mol Cancer Ther 2008, Kock et al. Neoplasia 2007,Sasportas et al. Proc Natl Acad Sci 2009, Pandita et al. GenesChromosomes Cancer 2004, Wakimoto et al. Cancer Res 2009, Baci-Onder etal., Cancer Res 2010). U87 human glioma cells were utilized in thisstudy for several reasons. The minimal invasiveness of U87 lead to solidintracranial tumors that was essential for resection of the primary masswhile allowing residual tumor cells to remain and drive recurrence.Further, the ability of NSC to track U87 (Ehtesham et al. Cancer Res2002, Jurvansuu et al., Cancer Res 2008) permitted assessment of NSCmigration out of the sECM towards GBM micro-deposits. In addition, thesensitivity of U87 to TRAIL-induced apoptosis (Hingtgen et al. MolCancer Ther 2008) was vital for determining the therapeutic potential ofsECM-encapsulated therapeutic stem cells. Lastly, the robust expressionof fluoresecent and bioluminescent transgenes following lentiviraltransduction by U87 (Hingtgen et al. Mol Cancer Ther 2008, Kock et al.Neoplasia 2007) was essential for the quantitative image-guidedresection and tracking the response or tumors to stem cell therapyreported in this study.

Despite extensive pre-clinical evidence demonstrating the potential ofcell-based therapy for GBM, no preclinical studies have explored methodsto introduce therapeutically “armed” stem cells in GBM resectioncavities. This is vital in order to prevent the stem cell “wash-out” andrapid diffusion from the resection cavity by CSF while allowing releaseof anti-tumor proteins directly into the resection cavity from thetransplanted stem cells. Biodegradable sECM formulations are anattractive approach for retention of stem cells in the resection cavity.Previous studies have shown that biodegradable sECM increase theviability of mNSC and their differentiation into neurons in vitro (Panet al. J Neurosci Res 2009). Recent in vivo studies illustratetransplanted biodegradable scaffolds containing stem (and otherneuronal) cells in models of degeneration and hypoxia-ischemia (Orive etal., Nat Rev Neurosci 2009).

In this study sECMs were employed that are based on a thiol-modifiedhyaluronic acid (HA) and a thiol reactive cross-linker (polyethyleneglycol diacrylate) which provides biocompatibility, physiologicalrelevance, and customizability (Xu et al. Prostaglandin Other LipidMediat 2009). Additionally, release profiles of sECM used in this studywere ideal to permit both migratory stem cells and secreted therapeuticproteins to exit the sECM. These events were confirmed by serialmonitoring of diagnostic markers (luciferase and fluorescence) whichrevealed extensive migration of mNSC out of sECMs towards GBM whilesecreting high levels of diagnostic proteins. sECM encapsulationdramatically increased the survival of mNSC in resection cavities ascompared to non-sECM encapsulated cells over a period of 4 weeks.Without wishing to be bound or limited by theory, the increased survivalof sECM encapsulated mNSC is related to the fact that encapsulation canenhance survival of transplanted cells by providing a physiologicallyrelevant environment that promotes attachment to reduce anoikis-mediateddeath, cell diffusion, and protection from the host immune system(Laflamme et al., Nat Biotechnol 2007, Mooney and Vandenburgh, Cell StemCell 2008, Terrovitis et al., J Am Coll Cardiol 2008, Zvibel et al.,Cell Transplant 2002). We demonstrate herein that sECM encapsulatedengineered mNSC are effective by way of increasing the concentration oftherapeutic stem cells at the site of tumor resection to extend the drugexposure time to tumor cells. As long-term survival of NSC in micebrains was critical in order to fully evaluate the therapeutic effectsof encapsulated therapeutic NSC, primary mouse NSC were utilized asopposed to human NSC.

The ability of TRAIL to selectively target tumor cells while remainingharmless to most normal cells makes it an attractive candidate for anapoptotic therapy for highly malignant brain tumors. However, sustainedlevels of TRAIL are key to improving the efficiency and potency ofTRAIL-based pro-apoptotic cancer therapy. It has been shown that mNSC donot express TRAIL receptors and are insensitive to TRAIL mediatedapoptosis. Using a diagnostic variant of S-TRAIL, the results shownherein clearly reveal that mNSC secreted S-TRAIL is released from sECMsand induces caspase-3 mediated apoptosis in brain tumor cells in vitro.When encapsulated into sECMs and implanted into resected GBM tumors,mNSC-S-TRAIL results in a significant increase in survival of micebearing GBMs. These results confirm that TRAIL is a potent inhibitor ofbrain tumor growth, and that encapsulated mNSC-S-TRAIL cytotoxic therapyis highly efficient in inducing apoptosis in residual GBM cells in ourmouse model of GBM resection. Furthermore, stem cells have the advantageof offering a continuous and concentrated local delivery of secretabletherapeutic molecules like TRAIL, thus reducing non-selective targeting,and allowing higher treatment efficiency and potency for a longer timeperiod. These results demonstrate that ECM encapsulation of mNSC-S-TRAILis a new approach for delivering cytotoxic therapies to GBM and have thepotential to improve patient outcomes.

Although TRAIL is a selective and potent anti-tumor agent, many tumorlines, including some established GBM lines, have varying resistance orsensitivity to TRAIL-induced apoptosis, with about 50% of alreadyestablished GBM lines being resistant to TRAIL(see FIG. 18, forexample). Although therapy with sECM-encapsulated TRAIL-secreting stemcells is efficacious for TRAIL-sensitive GBMs, many GBM tumors arelikely to be fully resistant to TRAIL-based therapies. To address GBMsthat are fully resistant to TRAIL, sensitizing TRAIL resistant GBM celllines to TRAIL-mediated apoptosis by treatment can be accomplished withagents including, for example, temozolomid, protease inhibitors,cisplatin, proteasome inhibitors, and daidzein. Recent in vivo studieshave shown that treatment with irradiation followed by TRAIL-secretingumbilical cord blood-derived MSCs synergistically enhances apoptosis inTRAIL-sensitive and TRAIL-resistant GBM. In addition, stem cells can beengineered to simultaneously secrete different therapeutic proteins thattarget multiple pathways in GBMs, in some embodiments. Along theselines, we have engineered stem cells to secrete an antiangiogenic agentconsisting of three type-1 anti-angiogenic repeats of thrombospondin-1(aaTSP-1) and shown in a recently published study that prolonged releaseof aaTSP-1 from stem cells in mice bearing gliomas targetsGBM-associated vasculature and increases mouse survival (Van Eekelen, M.et al. Human stem cells expressing novel TSP-1 variant haveantiangiogenic effect on brain tumors. Oncogene 29, 3185-3195 (2010)).As TSP-1 is known to normalize vasculature and upregulate deathreceptors DR4 and DR5 on tumor associated endothelial cells, the use ofstem cells expressing both TRAIL and TSP-1, in some embodiments, can beused to augment TRAIL-mediated apoptosis in both GBM cells andassociated endothelial cells.

The limited availability of non-invasive methods to monitor multiplemolecular events has been one of the main limitations in testing theefficacy of various tumor therapy paradigms. In previous studies, it hasbeen have shown that delivery of viruses (Shah et al. Oncogene 2003),NSC (Shah et al. Ann Neurol 2005) can be followed and GBM burden in vivocan be quanitified (Shah et al. Ann Neurol 2005, Kock et al. Neoplasia2007,Shah et al. Oncogene 2003) using non-invasive bioluminescenceimaging. However, combining bioluminescence imaging and confocalintravital microscopy offers great potential to image multiple events inreal time. As shown herein, tumor cells and stem cells can be labeledwith bimodal imaging markers (bioluminescent and fluorescent) expressedas a single transcript, which allows for expanding the number of eventsthat can be visualized in real-time in vivo and efficiently appliedbioluminescence imaging to follow both un-resected and resectedintracranial tumors and the fate of NSC in vivo. To followpharmacokinetics of therapeutic S-TRAIL both in culture and in mousemodels of GBM, a N-terminal fusion of S-TRAIL with Rluc(o) has beenemployed, which was engineered and characterized for its extracellularfunctionality. In addition, a blood pool agent, angiosense-750, has beenused to visualize the tumor associated vasculature. Clearly, theinclusion of non-invasive molecular imaging allows characterization ofmultiple events in sECM-NSC therapy with enhanced spatial and temporalresolution.

In recent years, primary GBM lines have been created from isolated humanbrain tumor tissue and used for preclinical studies. Several studieshave shown that xenografts of these primary cell lines oftenrecapitulate clinical GBM more faithfully than established GBM celllines, thus providing more insights and a more stringent test ofpromising new anti-GBM therapies. In developing the studies describedherein, we initially used an established glioma line, U87, that ourlaboratory and others have extensively characterized for in vivo tumorformation and assessment of target therapeutics in mouse models of braintumors. Most of the established glioma lines, including U87, form solidintracranial tumors in most cases. This was essential for resection ofthe primary mass and for study of the effect of sECMencapsulated mousestem cells on the residual tumor cells after resection. However, to testthe efficacy of sECM-encapsulated stem cell therapy in mouse models ofGBM that recapitulate all the features of the human cancer, we used theprimary cell line GBM8. We and others have shown GBM8 cells form highlyinvasive tumors upon intracerebral implantation into mic, thusrecapitulating most of the features of human GBM. Although primaryinvasive lines are more predictive, they introduce the technicaldifficulty of resecting invasive tumor cells from the brain. Tocircumvent this, we implanted ten times as many GBM8-mCherry-Fluc cellsas used by us in previous reports to create an initial solid tumor masswhich could then be resected. In addition to a more clinically relevanttumor model, we also used bone marrow-derived hMSCs that have beenextensively used in past and ongoing clinical trials and offer thepotential of autologous transplantation, thus overcoming the limitationof immune rejection. Pairing the clinically relevant GBM and hMSC linesallowed us to perform extensive studies investigating the effect ofencapsulated therapeutic hMSCs in invasive mouse model of GBM resection.Our results described herein clearly demonstrate that hMSC-S-TRAIL cellsrapidly attenuated progression of GBM8 tumors in the brains of mice.

Although most studies, including our previous studies in mouse models ofGBM, have shown that human bone marrow-derived MSCs offer the potentialof autologous transplantation and have antitumor therapeutic potential,some studies have suggested tumor-promoting properties in mouse tumormodel, and mass formation in an experimental autoimmuneencephalomyelitis model in mice. As the tumorigenic potential ofdifferent stem cell types is a concern, the use of alternative stem cellsources, including adipose tissue-derived MSCs49 and skin-derivedreprogrammed induced pluripotent NSCs (Mattis, V. B. & Svendsen, C. N.Induced pluripotent stem cells: a new revolution for clinical neurology?Lancet Neurol. 10, 383-394 (2011).) from patients, can be used in someembodiments of the compositions and methods described herein. In someembodiments, the incorporation of suicide genes, such as HSV-TK, intotherapeutic human stem cell types can be used to allow the eradicationof therapeutic stem cells after GBM treatment.

The studies shown herein reveal the fate and therapeutic efficacy ofengineered and sECM encapsulated mNSC in a mouse model of GBM resection.Using the compositions and methods described herein, advances can bemade in the way stem cells can be engineered and used clinically inbrain tumor patients. In some embodiments of the methods describedherein, neurosurgical removal of the main tumor mass at the time ofsurgery can be combined with implantation of patient's own reprogrammedcells or mesenchymal stem cells, therapeutically engineered withanti-tumor agent(s) and encapsulated in sECM, into the resection cavityof the tumor. These cells would result in killing of both residual andinvasive tumor cells with the ultimate goal of improving patientoutcomes. The results shown herein also have a major impact intranslating stem cell based therapies for the treatment of a number ofother brain pathologies.

Example 3—Additional Multimodal TRAIL Agents Path to Clinics UsingMultimodal Molecules

The HSV-TK (Herpes simplex virus-thymidine kinase) gene has beenexplored as a reporter and/or suicide gene previously. Both gene therapywith HSV-TK and the use of this gene as a marker are currently appliedin patients with various forms of cancer. Specifically, therapy has beenperformed by intratumoral injection of HSV-TK carriers (like viruses)followed by systemic ganciclovir injection and noninvasive positronemission tomography (PET) imaging of HSV-TK expression has beenperformed by using [¹⁸F]FHBG (a fluorine-18 labeled penciclovir analog)radiotracer.

Based on our multimodal TRAIL agent molecule, Rluc(o)TRAIL, describedherein is a multimodal molecule that can be used in clinical settings.Specifically, this can involve fusion of a secretable HSV-TK to theN-terminus of TRAIL to create a HSV-TK-TRAIL. This molecule can haveenhanced cancer cell killing properties because of the expression ofboth HSV-TK and TRAIL and in addition to diagnostic properties thatallow it to be tracked in mouse models of different cancers by eitherdelivering it systemically as a purified protein or via stem cells as asecretable protein.

Characterizing HSV-TK(L2)TRAIL in Culture and In Vivo

Generation of lentiviral vectors: Lentiviral vectors, pLV-CSC-IG bearingan IRES-GFP (Internal ribosomal entry site-green fluorescent protein)element can be used as a backbone and all restriction sites can beengineered into the primers. To generate fusion secretable HSV-TK andS-TRAIL fusion proteins, HSV-TK are initially PCR amplified with forwardprimers including the 60 bp signal sequence of Flt3-ligand usingLV-HSV-TK DNA as template. In addition S-TRAIL can be PCR amplifiedincluding signal sequence of Linker 2 (L2) (as described herein) as theforward primer and using LV-S-TRAIL as template. HSV-TK cDNA can beN-terminally fused to S-TRAIL cDNA and cloned into pLV-CSC-IG, thusresulting in a LV-HSV-TK(L2)TRAIL construct. This lentiviral constructcan be packaged into virions in 293T/17 cells using a helper virus-freepackaging system as described previously.

Characterization of LV-HSV-TK(L2)-TRAIL in vitro and in vivo: All theexperiments designed for Rluc(0)TRAIL fusion protein described hereincan be performed. In this study, the bioluminescence imaging, asdescribed herein can be replaced with PET imaging both in vitro and invivo which is described below.

In vitro assay: The activity of the TK fusion proteins in the crude celllysates of mammalian cells (293T and NSC expressing HSV-TK(L2)TRAIL canbe determined using [18F]FHBG as a substrate. Since cell culture mediummay contain free thymidine, all samples for enzyme activity assays canbe pre-treated with 80 units thymidine phosphorylase for 30 min at 378C,to hydrolyze any thymidine that could inhibit the TK reaction with[18F]FHBG. TK activity will be determined by incubating [18F]FHBG in thereaction mixture at 378C (0.17 nM, specific activity, 54,000 GBq/mmol).Samples of this mixture are loaded on a Whatman DE-81 filter atdifferent time points 25 ml. The negatively charged phosphorylated[18F]FHBG can be bound to these filters. Each filter will be washedthree times and radioactivity of the filters can be counted with a gammacounter. At the end of the experiment, 50 ml of reaction mixture can beloaded on a filter and the activity of this filter is measured withoutwashing (reflects both unchanged and phosphorylated [18F]FHBG). Theunwashed filter can be used to normalize the activity bound on thewashed filters. [18F]FHBG phosphorylation can be calculated by dividingthe radioactivity (cpm) of the washed filters by the radioactivity ofthe unwashed filters.

In vivo PET imaging: Mice bearing tumors can be either injectedintravenously with purified HSV-TK(L2)TRAIL or NSC expressingHSV-TK(L2)TRAIL as described for Rluco(L2)TRAIL and in vivo PET imagingcan be performed after the intravenous administration of 18F-FHBG (3764MBq) given 24 hrs after HSV-TK(L2)TRAIL administration. PET can beperformed at 3 h after the intravenous administration of the label. Twoemission images of 10 min each can be acquired at 30 min and 4 h later.After acquisition of the emission data, a transmission scan can beperformed in a single mode using a rotating 370-MBq 137Cs source placedoutside the field of view. To determine the site of the 18F-FHBGactivity, color-coded coronal emission images can be superimposed oninverted gray-scaled transmission images. PET imaging can be performedevery week for a period of 1 month. After each imaging session, the micecan be euthanized and their brains harvested and placed into a wellautomatic g-counter (1480 Wizard 3″; Wallac/PerkinElmer).

Assessing therapeutic efficacy of HSV-TK(L2)TRAIL in vivo: In order tocompare the therapeutic efficacy of TRAIL and HSV-TK(L2)TRAIL in micebearing gliomas, mice can be implanted in NSC expressing either S-TRAILor HSV-TK(L2)TRAIL and changes in tumor volumes can be followed by Flucbioluminescence imaging as described herein.

Materials and Methods Generation of Lentiviral Vectors

Lentiviral vectors, pLV-CSC-IG bearing an IRES-GFP (Internal ribosomalentry site-green fluorescent protein) element (Sena-Esteves et al JVivol Methods 2004), were used as a backbone and all restriction siteswere engineered into the primers. To generate the fusions LV-TRFL,LV-TRRL, and LV-TRGp, S-TRAIL and luciferases were PCR amplified usingLV-S-TRAIL (Shah et al Cancer Res. 2004), LV-GFP-Fluc (Shah et al.2008), LV-RLuc-DsRed2 (Shah et al. 2008), or pGLuc-Basic (New EnglandBiolabs, Ipswich Mass.) respectively as templates. The resulting S-TRAILfragment was digested with NheI and BamHI, while luciferase fragmentswere digested with BamHI/XhoI. Both fragments were ligated in-frame intoNheI/XhoI digested pLV-CSC-IG vector. LV-GpTR, LV-GpL₁TR, and LV-GpL2TRwere created by PCR amplification of GpLuc and digestion with NheI andEcoV. The S-TRAIL fragment was PCR amplified and 2, 6, or 12 amino acidlinkers were incorporated into the S-TRAIL forward primer. The resultingS-TRAIL fragment was digested with EcoV/XhoI and both fragments ligatedinto NheI/XhoI digested pLV-CSC-IG vector. To generate fusion proteinswith altered signal sequence GpLuc or RLuco fragments were PCR amplifiedwith forward primers including the 60 bp signal sequence of Flt3-ligandand reverse primer containing EcoV and XhoI restriction sites usingpGLuc-Basic (New England Biolabs) or optimized RLuc as templates. Thefragments were digested NheI/XhoI and ligated into NheI/XhoI digestedpLV-CSC-IG vector resulting in LV-SGpLuc and LV-SRLuco. LV-SGpL2TR andLV-SRL_(O)L₂TR were created inserting an S-TRAIL fragment containing L2digested with EcoV/XhoI into the LV-SGp or LV-SRLo vectors digested withEcoV/XhoI. All lentiviral constructs were packaged as lentiviral vectorsin 293T/17 cells using a helper virus-free packaging system as describedpreviously(Kock et al Neoplasia 2007). Briefly, lentiviral vectors wereproduced by transient transfection of 293T cells. Cells (15×10⁶) wereseeded in 150 mm² tissue culture plates 24 hrs before transfection inDMEM with 10% FBS, and cells were washed with fresh medium 4 hrs beforetransfection. Transfection was performed by calcium phosphateprecipitation method using 18 pg of transfer plasmid DNA, transfervectors constructed above, and the lentiviral helper plasmids pCMVΔ8.91(18 μg) and glycoprotein expression plasmid pVSVG (12 μg; Clontech).Cells were washed with fresh medium 16-18 hrs after transfection, andvector supernatants were harvested 48 hrs after transfection. Thesupernatants were filtered (0.45 μm) and loaded in a Beckman Quick-Sealultracentrifuge tube (Beckman Coulter, Fullerton, Calif.) andcentrifuged at 28,000×g for 90 min. Pellets were resuspended in PBS andstored at −80° C. Titers were determined by counting fluorescenttransduced 293T cells.

In Example 2, the following lentiviral vectors were used: LV-GFP,LV-GFP-Fluc, LV-GFP-Rluc, LV-Fluc-DsRed2 (Shah et al. J Neurosci 2008),Pico-mCherry-Fluc, LV-Ss-Rluc (o), LV-S-TRAIL (Kock et al. Neoplasia2007) and LV-Di-S-STRAIL. Both LV-S-TRAIL and LV-Di-S-TRAIL has anIRES-GFP (Internal ribosomal entry site-green fluorescent protein)element in the backbone. All lentiviral constructs were packaged aslentiviral vectors in 293T/17 cells using a helper virus-free packagingsystem. Stem cells and GBM cells were transduced with LVs at varyingmultiplicity of infection (MOI) by incubating virions in a culturemedium containing 4 μg/ml protamine sulfate (Sigma) and cells werevisualized for fluorescent protein expression by fluorescencemicroscopy. Following expansion in culture, both stem cells and GBMcells were sorted by fluorescence activated cell sorting or flowcytometry (FACSAria Cell-Sorting System, BD Biosciences). The effects ofdifferent concentrations of S-TRAIL on established and primary GBM cellviability were measured using an ATP-dependent luminescence reagent(CellTiterGlo, Promega).

Cell Lines and Cell Culture

U251, Gli36-EGFRvIII, Gli36-EGFRvIII-FD human glioma cells and 293Tcells were grown as described previously (Shah et al. 2008, Kock et alNeoplasia 2007). Primary mouse neural stem cells (Stem CellTechnologies, Vancouver. BC, Canada) were grown as previously described(Reynolds and Weiss, Dev. Biol., 1996) in Neurocult® NSC Basal media(Stem Cell Technologies) supplemented with proliferation supplements(Stem Cell Technologies) and EGF (20 ng/mL, R&D Systems, Minneapolis,Minn.). Adherent cultures were established by culturing mNSC on tissueculture plates coated with laminin and poly-ornithine (Sigma, St. Louis,Mo.). 293T, mNSC, and glioma cells were transduced with LVs byincubating virions in a culture medium containing 4 μg/ml protaminesulfate (Sigma).

Western blot analysis, ELISA, Viability, Caspase 3/7 activation: 24 hafter infection of 293T, treatment of glioma cells with media containingluciferase/S-TRAIL fusions, or non-treated mNSC and glioma cells, cellswere collected in NP-40 lysis buffer and equal amounts of protein (30μg) were denatured, separated by SDS-PAGE, transferred to nitrocellulosemembrane, blocked, and incubated over night at room temperature withantibodies against TRAIL (R&D Systems, Minneapolis, Minn.), DR4 (Sigma),human caspase-8 (Cell Signaling Technologies, Danvers, Mass.), orcleaved PARP (Cell Signaling Technologies). After incubation, the blotswere washed and further incubated for 1 h with peroxidase-conjugatedsecondary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) inTBS-T. Blots were developed using enhanced chemiluminescence reagents(Amersham, Piscataway, N.J.). Membranes were then exposed to film for 30s to 30 min, and quantified using NIH Image (National Institute ofHealth, Bethesda, Md.).

In Example 2, human glioma lines U87, LN229, A172, U251, Gli36vIII,Gli79, LN319 and U87 and primary GBM8-EF, GBM4, GBM6, GBM18 and BT74cells were grown. Primary mouse neural stem cells were grown in NSCBasal medium (Stem Cell Technologies) supplemented with proliferationsupplements (Stem Cell Technologies) and epidermal growth factor (20 ngml-1, R&D Systems). Adherent cultures were established by culturingmNSCs on tissue culture plates coated with laminin and poly-ornithine(Sigma). Human bone marrow-derived MSCs were grown as describedpreviously.

Human GBM cells, U87 were incubated with conditioned medium from sECMencapsulated mNSC expressing S-TRAIL or Ss-Rluc(o) for 18 hrs, lysed andcentrifuged at 30,000 g for 30 min at 4° C. Equal amounts of total cellprotein (30 μg) were denatured, separated by SDS-PAGE, and transferredto nitrocellulose membrane, blocked and incubated for 1 h at roomtemperature with rabbit polyclonal antibodies to proteins: (a) cleavedPARP and (b) caspase-8 (Cell Science, MA). Blots were developed usingenhanced chemiluminescence reagents (Amersham). Membranes were thenexposed to film for 30 s to 30 min.

For ELISA, eighty-percent confluent 6-well dishes of transduced 293Tcells were washed with PBS and incubated in 3 mL OptiMEM(Gibco/Invitrogen, Carlsbad, Calif.) for 24 h. S-TRAIL concentration inthe conditioned culture medium was measured by ELISA with the TRAILImmunoassay Kit (Biosource International, Camarillo, Calif.) accordingto the manufacturer's protocol, using recombinant human TRAIL expressedin Escherichia coli as a standard.

To determine the effects of S-TRAIL fusion proteins on glioma cells,Gli36-EGFRvIII cells (0.7×10⁶) were incubated with conditioned mediafrom 293T equally transduced with LV vectors encoding luciferase/S-TRAILfusion proteins. Twenty-four hours after treatment, remaining cells werewashed and resuspended in 500 μl of media. The metabolic activity of thecells was determined using a luminescent adenosine triphosphate(ATP)-based assay (CellTiter GLO; Promega, Madison, Wis.) on 25 μl ofcells from the suspension according to the manufacturer's instructions.Additionally, caspase 3/7 activation was determined using theluciferase-based Caspase GLO assay (Promega) on 25 μl of cells from thesuspension according to the manufacturer's instructions.

To investigate the effects of TRAIL on mNSC viability, mNSC (0.1×10⁶cells) were treated with increasing concentrations (0-100 ng/mL) ofpurified S-TRAIL for 24 hours. After incubation, cells were collected in500 μl, and cell viability was determined using 50 μl of the cellsuspension as described above.

Bioluminescent Imaging of S-TRAIL Fusion Activity In Vitro

For imaging of S-TRAIL-luciferase fusion protein secretion, culturemedia was collected from 293T or different stem cell lines transducedwith LV encoding various fusion proteins. The culture medium containingsecreted fusion proteins or the cells were collected 24 h afterrefreshing. The luciferase activity in both cells and medium wasdetermined by incubating the medium or cells with 1 μg/ml coelenterazinefor GpLuc- and RLuc-containing fusions or 150 μg/ml D-luciferin for theFLuc-containing fusion, and imaged for 1 min using a cryogenicallycooled high efficiency CCD camera system. Post processing andvisualization were performed as described previously (Shah et al., AnnNeurol, 2005).

To determine the correlation between the number of transduced cells andthe bioluminescence signal, cells were seeded in differentconcentrations and substrates for luciferases (1 μg/ml coelenterazinefor RLuc and GpLuc; 1.5 μg/ml D-luciferin for FLuc) were added to themedium. Luciferase activity was measured using a luminometer at 1sec/well. To determine the duration of transgene expression, differentstem cell lines were transduced with SRL_(O)L2TR. On days 0, 2, 7, and14 post-transduction, cells (1×10⁵ cells) were collected, combined withcoelenterazine (1 μg/ml), and luciferase activity was determined using aluminometer.

To determine relative levels of SRL_(O)L2TR secretion, different stemcell lines were transduced with LV-SRL_(O)L₂TR and plated at increasingconcentrations in 96 well plates. 24 hrs after transduction, equalvolumes of media were collected, mixed with coelenterazine (1 μg/ml),and luciferase activity was determine using a luminometer.

For co-culture experiments different stem cell lines were transducedwith appropriate MOI. 48 hrs after transduction, stem cells were platedat increasing numbers (0-1.5×10⁴) in 96 well plates. 24 hours afterseeding stem cells, Gli36-EGFRvIII or U251 cells (5×10³) expressingmCherryFluc were overlayed on the seeded stem cell. After co-culture for24 hours, media was transferred to a new plate, combined withcoelenterazine (1 μg/ml) and imaged to visualize levels of SRL_(O)L2TR.At the same time, 1.5 μg/ml D-luciferin for FLuc was added to theremaining glioma cells and glioma cell viability was determined bybioluminescence imaging and measuring in a luminometer.

Bioluminescent Imaging of S-TRAIL Fusion Activity In Vivo

In Example 1, to monitor the pharmacokinetics of S-TRAIL secretion invivo, SCID mice (SCID; 3 weeks of age; Charles River Laboratories) wereimplanted with 4×10⁶ U251-FD, U251-FD-SGpL2TR, or U251-FD-SRL_(O)L₂TRsubcutaneously in SCID mice (n=4). Mice were then serially imaged forGpLuc or RLuc activity on day 1, 3, and 15 by injecting mice with 100 μgof coelenterazine imaged photon emission determined using acryogenically cooled high efficiency CCD camera system. To determinetumor volume, mice were injected with 2 mg D-luciferin and FLuc imagingwas performed on day 2, 7, and 15. To simultaneously visualize tumorsize and release of GpLuc or RLuc with modified secretion sequence, U251glioma cells were transduced with LV encoding FlucDsRed2, followed byinfection with equal MOI of either LV-SGpLuc or LV-SRLuco. 5×10⁶ cellswere implanted in duplicate subcutaneously in SCID mice (n=6). GpLuc andRLuc imaging were performed to visualize secretion of modified proteinson days 1, 5, and 10 as described above, followed 8 hours later by Flucimaging to visualize tumor size as above.

In Example 1, to investigate the kinetics of tumor localization, micewere implanted with 4×10⁶ Gli36-EGFRvIII-FD. Twenty-four hours later,mice were injected with 2 mg D-luciferin and FLuc imaging was performedto identify tumor location. Twenty-four hours later, mice were injectedwith media containing SRL_(O)L₂TR by either intravenous infusion ordirect injection around the established tumor. Five minutespost-injection, mice were injected with 100 μg of coelenterazine andimaged every 5 minutes for 40 minutes, and at 24 hours post-injection.In a separate set of mice, animals were sacrificed 1 hour post-mediainjection, the liver, lung, tumor kidney, urine, and blood werecollected and imaged ex vivo. The tissue was weighed and data expressedrelative to tissue weight. In a separate set of mice, FLuc imaging wasperformed 48 hours post-media injection to determine the effects ofSRL_(O)L2TR on subcutaneous tumor growth.

In Example 2, to simultaneously visualize survival of encapsulated mNSCand release of Ss-Rluc(o), mNSC expressing GFP-Fluc and Ss-Rluc(o) wereimplanted in the frontal lobe of nude mice (n=6) as described above. Fordual-luciferase imaging 7, 14, 21 and 28 hours after implantation, micewere injected with 100 ug of coelenterazine/mouse via tail vein andimaged for Ss-Rluc (o) activity as described previously (Shah et al. AnnNeurol 2005). Eighteen hours later, when there was no residualcoelenterazine/Rluc activity, mice were injected with 1 mgD-luciferin/mouse intraperitoneally and imaged for Fluc activity after 5minutes, as described above. Post processing and visualization wereperformed as described previously (Shah et al. Ann Neurol 2005) Tosimultaneously visualize tumor volumes and caspase-3/7 activity, micebearing U87-mCherry-Fluc tumors were resected and implanted withencapsulated mNSC-TRAIL or mNSC-GFP-Rluc in the resection cavity. Tumorvolumes were followed by imaging mice for Fluc activity as describedabove. For imaging apoptosis induced by S-TRAIL expression, mice weregiven an intraperitoneal (i.p.) injection of highly purified Caspase-Glo3/7 reagent (Promega, Madison, Wis.; 5 mg in 150 μL DMSO) and imaged forcaspase-3 dependent luciferase activity for 5 minute afteradministration of Caspase-Glo 3/7 reagent. Post processing andvisualization were performed as described previously (Shah et al. AnnNeurol 2005).

Immunocytochemistry and Immunohistochemistry

To assess the differentiation potential of cultured mNSC, cells wereplated on laminin/polyornithine coated coverslips and incubated inNeurocult media containing differentiation supplements (Stem CellTechnologies) for 10 days. Following differentiation, cells were washed2× with PBS and fixed with 4% paraformaldehyde for 20 minutes. The cellswere then permeabilized and incubated with anti-Nestin (Millipore,Billireca, Mass.), anti-GFAP (Chemicon), and anti-Olig2 (Chemicon)antibodies for 1 hour at 37° C. The cells were washed and incubated withAlexa dye 555 nm (Invitrogen) for 1 hour, washed, mounted, and examinedmicroscopically.

To investigate the differentiation potential of SRL_(O)L₂TR-secretingmNSC, a separate cohort of mice were implanted with a mix of mNSCexpressing SRL_(O)L₂TR and unlabelled Gli36-EGFFvIII glioma cells. Onday 4 post-implantation, mice were perfused, brains were removed, and 30μM sections of the brains were generated using a vibratome. Floatingbrain sections were immunostained with antibodies against mouse nestin,GFAP, Tuj-1 (Covance, Princeton, N.J.), and Ki67 (DAKO, Carpinteria,Calif.), followed by incubation with Alexa dye 555 nm secondaryantibodies, mounting, and visualization by confocal microscopy.

To investigate the migration of transduced mNSCs towards gliomas,Gli36-EGFRvIII-FD were stereotactically implanted into the frontal lobeof mice. On day 3 post-implantation, transduced mNSC expressing GFP-RLucwere implanted 1 mm lateral to the gliomas. Mice were sacrificed 2, 5,and 10 days after mNSC implantation. The brains were collected,sectioned, mounted, and cells expressing fluorescent markers werevisualized using confocal microscopy.

Tissue Processing

Mice bearing tumors in the cranial window or mice with resected tumorsor mice with resected tumors and implanted with sECM encapsulated mNSCwere perfused with formalin and brains were removed and sectioned. Thetissue sections were dehydrated in xylene and ethanol, immersed in PBSand stained with hematoxylin and Eosin (H&E). Photomicrographs of bothIHC and H&E slides were taken using the Nikon E400 light microscope(Nikon Instruments Inc, Melville, N.Y.) attached to a SPOT CCD digitalcamera (Diagnostics Instruments, Inc., Sterling Heights, Mich.)

sECM Encapsulated mNSC: Cell Viability, ELISA and Release of SecretedProteins:

The sECM components, Hystem and Extralink (Glycosan Biosystems, SaltLake City, Utah) were reconstituted according to the protocol providedby Glycosan Biosystems. Specifically, 1, 2 or 4×10⁵ mNSC expressingeither a) GFP-Fluc and Ss-Rluc (o) orb)S-TRAIL were resuspended inHystem (11 μl) and Extralink (9 μl) was added to cross-link the matrixas described in the protocol. After 20 minutes (gelation time) at roomtemperature (25 C), the mNSC-sECM hydrogel was placed in the center ofdifferent sizes (35 or 60 mm) of glass-bottom dishes.

To determine the correlation between the number of U87-Fluc-mCherry orsECM encapsulated mNSC expressing GFP-Fluc and Ss-Rluc(o) andbioluminescence signal, different numbers of mNSC were encapsulated insECMs and U87-mCherry-Fluc cells were seeded in different concentrationsand D-luciferin (1.5ug/ml) was added to the medium and luciferaseactivity was measured using a cryogenically cooled high efficiency CCDcamera system (Roper Scientific, Trenton, N.J., USA). Each experimentwas performed in triplicate. To simultaneously assess mNSC viability andrelease of mNSC-secreted proteins out of sECM, mNSC expressing GFP-Flucand Ss-Rluc(o) were encapsulated into sECM and followed by Fluc and Rlucbioluminescence imaging in vitro as described herein over a period of 12days. To assess TRAIL concentration, the conditioned medium fromsECM-mNSC expressing Ss-Rluc(o) or S-TRAIL was collected and ELISA wasperformed as described previously (Kock et al. Neoplasia 2007).

Statistical Analysis

Data were analyzed by Student t test when comparing 2 groups and byANOVA, followed by Dunnetts post-test when comparing greater than 2groups. Data were expressed as mean±SEM and differences were consideredsignificant at P<0.05. Survival times of mice groups were compared usinglogrank test.

Intracranial mNSC Survival and Tumor Progression

To assess the survival of transduced mNSC in the brain, 1×106 mNSCtransduced with LV-GFP-FLuc were mixed with Gli36-EGFRvIII (0.1×106) andstereotactically implanted (from bregma, ML: 2.5 mm, SI: 2 mm) (n=4 ineach case) in mice as described previously (Shah et al. 2008). FLucimaging was performed 2, 6, 9, and 12 days post-injection by giving miceintraperitoneal injection of 2 mg of D-luciferin and collecting photonemission over 5 minutes with a cooled charge-coupled device camera.Images were processed as described previously (Shah et al. 2008). Todetermine the effects of SRLOL2TR on intracranial gliomas,Gli36-EGFRvIII-FD and mNSC expressing SRLOL2TR were harvested at 80%confluency and a mix of glioma cells (5×105) and transduced mNSC (1×106)(n=4 in each case) was implanted stereotactically (from bregma, ML: 2.5mm, SI: 2 mm) (n=4 in each case). Mice were injected intraperitoneallywith 4.5 mg/mouse of D-luciferin on days 1, 3, 6, 9, 13, and 21 andimaged as described above. To monitor SRLOL2TR from mNSC, the same micewere imaged for RLucO activity on days 2, 6, 9, and 12 by injecting 100μg of coelenterazine intravenously, and 5 minutes later photon emissionwas determined over 7 minutes. All images were processed as describedpreviously (Shah et al. 2008).

Creation of a Mouse Model of Resection

Athymic nude mice (6-8 weeks of age; Charles River Laboratories,Wilmington, Mass.) 25-30 g in weight (n=8 in each group) were used forthe intracranial xenograft GBM model. One week prior to GBM cellimplantation, mice were immobilized on a stereotactic frame and a smallcircular portion of the skull (˜7 mm diameter) was removed, and the durawas gently peeled back from the cortical surface. U87 expressingFluc-mCherry were harvested at 80% confluency and implantedstereotactically (7.5×10⁴ or 1.5×10⁵ (n=16 in each case) in the rightfrontal lobe and the skin was sutured together. On the day of tumorresection (day 14: for mice implanted with 7.5×10⁴ tumor cells; and day21: for mice implanted with 1.5×10⁵ cells post tumor cell implantation;n=16 in each group), mice were imaged by Fluc bioluminescence imaging(n=16 from each group) and intravital microscopy (n=3 in each group) asdescribed previously (Shah et al. J Neurosci 2008, Arwent et al. CancerRes 2007). For intravital microscopy, angiosense-750 (Visen Medical) wasinjected and mice were imaged to visualize tumor volumes and associatedvasculature as described previously (Van Eekelen et al. Oncogene 2010).For tumor resection, mice (n=8 from each group) were immobilized on astereotactic frame, the skin was opened and the superficial tumor wasexposed. A dissecting Leica surgical microscope with 20× magnificationwas used for mechanical resection to reduce the tumor volume up to thetumor-tissue interface, leaving margins of the dura intact. Finally, thewound was copiously irrigated and the skin closed with 4-0 Vicrylsuture. Mice were imaged by bioluminescence imaging and intravitalmicroscopy post-resection. Ex-vivo analysis on resected tumors wasperformed by incubating tumors with 1.5 μg/ml D-luciferin in PBS and bybioluminescence imaging. The studies described herein used 140 femalemice.

In Vitro Imaging of S-TRAIL Secretion, Tumor Cell Viability andCaspase3/7 Activity

In Example 2, mNSCs expressing Ss-Rluc(o) or S-TRAIL (1×10⁵) wereencapsulated in sECM and placed in a 35 mm plate as described above.Human GBM cells, U87-mCherry-Fluc (2×10⁵) were plated around thesECM-NSC and cell viability at different time points (8-24 hours) wasmeasured by quantitative in vitro bioluminescence imaging as describedherein. GBM cells were also assessed at different time points (8-24hours) for caspase-3/7 activity with a caged, caspase 3/7-activatableDEVD-aminoluciferin (Caspase-Glo 3/7, Promega, Madison, Wis., USA) asdescribed previously (Shah et al. Ann Neurol 2005). For co-cultureexperiments, increasing numbers (0-4×10⁵) of mNSC expressing Di-S-TRAILwere encapsulated in sECM, plated and 24 hrs later, U87-mCherry-Fluc(1×10⁵) were seeded around the sECM encapsulated mNSC. After co-culturefor 24 hrs, media was transferred to a new plate, combined withcoelenterazine (1 μg/ml) and imaged to visualize levels of Di-S-TRAIL.At the same time, 1.5 μg/ml D-luciferin for FLuc was added to theremaining GBM cells and GBM cell viability was determined bybioluminescence imaging and measuring in a luminometer. Similarly, hMSCsexpressing GFP or S-TRAIL (1×10⁵) were encapsulated in sECM and placedin a 35-mm plate, GBM8-mCherry-Fluc cells (2×10⁵) were plated around thesECM-encapsulated hMSCs, and cell viability and caspase3/7 activity weremeasured by quantitative in vitro bioluminescence imaging at differenttime points (15-24 h) as described herein above.

sECM-mNSC Survival and Migration Studies:

In Example 2, to compare sECM encapsulated and non-encapsulated mNSCsurvival in athymic nude mice, GFP-Fluc expressing mNSC (5×10⁵/mouse)were either resuspended in PBS or encapsulated in sECMs and implantedstereotaxically (n=5 in each case) in the right frontal lobe (frombregma, AP: −2 mm, ML: 2 mm V (from dura):2 mm) and mice were imaged onweeks 1-4 for Fluc activity as described below. To compare sECMencapsulated and non-encapsulated mNSC survival in the tumor resectioncavity, mice implanted with U87-Rluc-DsRed2 tumors in the cranial windowwere imaged for Rluc activity by injecting 100 ug ofcoelenterazine/mouse via tail vein (described in detail herein),resected and implanted with GFP-Fluc expressing mNSC (5×10⁵/mouse)either resuspended in PBS or encapsulated in sECM (n=5 in each case). Toencapsulate mNSC expressing GFP-Fluc, mNSC were resuspended with the twodifferent components of sECM as described above and 10 min later, themNSC-sECM encapsulated mix was placed in the resection cavityimmediately after tumor resection and the skin was closed with 4-0Vicryl suture. Mice were imaged on week 1-4 for Fluc activity asdescribed below and intravital microscopy as described above. To studythe migration of sECM encapsulated mNSC toward GBMs, U87-mCherry-Fluccells (5×10⁴) were implanted stereotaxically into the right frontal lobein cranial windows of nude mice (n=10) and 7 days later, encapsulatedmNSC-GFP-Fluc (n=5) or saline (n=4) were injected into the right frontallobe of tumor-bearing mice. mNSC migration was followed by intravitalmicroscopy as described earlier (Shah et al. J Neurosci 2008).

To study the effect of therapeutic mNSC-S-TRAIL encapsulated in sECM inthe resection model, mice (n=32) bearing U87-mCherry-Fluc GBMs in thecranial window were imaged for Fluc activity on the day of resection(Day 21 post tumor cell implantation), divided into 4 groups (n=8 ineach group) by distributing mice of matching tumor sizes (indicated bythe Fluc signal intensity) equally across all groups, and resected(n=24) as described above. Mice were immediately imaged for Fluc signalintensity post-resection. sECM encapsulated mNSC-S-TRAIL (n=8),mNSC-GFP-Rluc (n=8) or non-encapsulated mNSC-S-TRAIL (n=8) were placedin the tumor resection cavity and mice were followed for survival overtime (including un-resected controls, n=8). Tumor volumes in themNSC-S-TRAIL and mNSC-GFP-Rluc group were imaged by Fluc bioluminescenceimaging as described earlier (Sasportas et al. Proc Natl Acad Sci 2009).To study the effects of therapeutic hMSC-S-TRAIL cells encapsulated insECM in the primary invasive GBM8 resection model, mice (n=14) bearingGBM8-mCherry-Fluc tumors in the cranial window were imaged for Flucactivity on the day of resection (day 7 after tumor cell implantation)and divided into two groups (n=7 in each group) as described above.sECM-encapsulated hMSC-S-TRAIL (n=7) or hMSC-GFP (n=7) cells were placedin the tumor resection cavity and tumor volumes were imaged by Flucbioluminescence imaging.

Western Blot Analysis.

In Example 2, human U87 GBM cells were incubated with conditioned mediumfrom sECM-encapsulated mNSCs expressing S-TRAIL or Ss-Rluc(o) for 18 h,lysed and centrifuged at 30,000 g for 30 min at 4° C. Equal amounts oftotal cellular protein (30 μg) were denatured, separated by SDS-PAGE,transferred to nitrocellulose membrane, blocked and incubated for 1 h at25° C. with rabbit polyclonal antibodies to cleaved PARP and caspase-8(Cell Science). Blots were developed using enhanced chemiluminescencereagents (Amersham). Membranes were then exposed to film for 30 s to 30min.

Collagen Invasion Assay

In Example 2, human GBM8-Fluc-mCherry cells created from the GBM8-EFline were grown as spheres and resuspended in rat tail collagen, type 1(BD Biosciences). The solution was then allowed to solidify in a culturedish and the cells were supplemented with the growth medium.Collagen-embedded GBM8-Fluc-mCherry cells were imaged at days 0 and 5 tovisualize dispersal of GBM8-Fluc-mCherry cells into the collagen matrixfrom the primary sphere.

Dual Bioluminescence Imaging In Vivo.

In Example 2, to simultaneously visualize survival of encapsulated mNSCsand release of Ss-Rluc(o), mNSCs expressing GFP-Fluc and Ss-Rluc(o) wereimplanted in the frontal lobe of nude mice (n=6) as described hereinabove. For dual luciferase imaging 7, 14, 21 and 28 d afterimplantation, mice were injected with 100 μg of coelenterazine per mousethrough the tail vein and imaged for Ss-Rluc(o) activity. Eighteen hourslater, when there was no residual coelenterazine-Rluc activity, micewere injected with 1 mg d-luciferin per mouse intraperitoneally andimaged for Fluc activity 5 min later as described herein above.Postprocessing and visualization were performed. To simultaneouslyvisualize tumor volumes and caspase-3/7 activity, mice bearingU87-mCherry-Fluc tumors were resected and implanted with encapsulatedmNSC-TRAIL or mNSC-GFP-Rluc cells in the resection cavity. Tumor volumeswere followed by imaging mice for Fluc activity as described hereinabove. For imaging apoptosis induced by S-TRAIL expression, mice wereinjected intraperitoneally with highly purified Caspase-Glo 3/7 reagent(5 mg in 150 μl DMSO) and imaged for caspase-3-dependent luciferaseactivity for 5 min after administration of the Caspase-Glo 3/7 reagent.Postprocessing and visualization were performed. All images are thevisible light image superimposed with bioluminescence images with ascale in photons min⁻¹ cm⁻².

Tissue Processing

Mice bearing tumors in the cranial window or mice with resected tumorsor mice with resected tumors and implanted with sECMencapsulated mNSCsor hMSCs were perfused with formalin and brains were removed andsectioned. Cleaved caspase-3 immunohistochemical staining on brainsections was performed. Photomicrographs of immunohistochemistry andhematoxylin and eosin slides were taken using a Nikon E400 lightmicroscope attached to a SPOT CCD digital camera (DiagnosticsInstruments).

Statistical Analysis.

Data were analyzed by Student t-test when comparing two groups and byANOVA, followed by Dunnett's post-test, when comparing more than twogroups. Data are expressed as mean s.e.m., and differences wereconsidered significant at P<0.05. Survival times of groups of mice werecompared using a log-rank test.

TABLE 1 Composition and acitivity of luciferase and S-TRAIL fusionproteins: − (no activity); ++++ (highest activity) Ther- a- Diagnosticpeutic Extra- In In In cellular Vivo Vivo Vivo Construct In Vivo Activ-Activ- Activ- Name Fusion Activity ity ity ity TRRL S-TRAIL-RLu 

− N/A − N/A FRFL S-TRAIL-Fla 

− N/A − N/A RRG 

S-TRAIL-GpLuc ++ N/A − N/A GpTR CpLue-S-TRAIL + N/A + N/A GpL₁TRGpPLuc-L1-S-TRAIL ++ N/A + N/A GPL₂TR GpLuc-L3-S-TRAIL +++ N/A +++ N/ASGpL₂TR S-GpLic-L2-S-TRAIL ++++ + ++++ ++++ SRL₀I₀TR S-RLuc-L2-S-TRAIL+++ ++++ +++ ++++

indicates data missing or illegible when filed

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1.-22. (canceled)
 23. A composition comprising a T cell comprising anucleic acid encoding a polypeptide comprising a soluble TRAIL fusionprotein comprising a reporter module, a linker module, and a therapeuticTRAIL module comprising an extracellular domain of human TRAIL of SEQ IDNO:
 1. 24. The composition of claim 23, wherein the cell is encapsulatedin a matrix or scaffold.
 25. The composition of claim 23, wherein thetherapeutic TRAIL module comprises amino acids 39-281 of SEQ ID NO: 1.26. The composition of claim 23, wherein the therapeutic TRAIL modulecomprises amino acids 95-281 of SEQ ID NO:
 1. 27. The composition ofclaim 23, wherein the therapeutic TRAIL module comprises amino acids114-281 of SEQ ID NO:
 1. 28. The composition of claim 23, wherein thetherapeutic TRAIL module consists of amino acids 114-281 of SEQ IDNO:
 1. 29. The composition of claim 24, wherein the matrix comprises asynthetic extracellular matrix.
 30. The composition of claim 24, whereinthe matrix is biodegradable.
 31. The composition of claim 29, whereinthe synthetic extracellular matrix comprises a thiol-modified hyaluronicacid and a thiol-reactive cross-linker molecule.
 32. The composition ofclaim 30, wherein the thiol-reactive cross-linker molecule ispolyethylene glycol diacrylate.
 33. The composition of claim 23, furthercomprising a nucleic acid sequence encoding HSV-TK.
 34. The compositionof claim 23, wherein the polypeptide comprises, in the followingN-terminal to C-terminal order, a reporter module, a linker module of atleast 8 amino acids, and a therapeutic TRAIL module comprising anextracellular domain of human TRAIL of SEQ ID NO:
 1. 35. The compositionof claim 34, wherein the linker domain comprises the amino acid sequenceof SEQ ID NO:
 4. 36. A composition comprising a T cell comprising aheterologous nucleic acid sequence encoding a soluble TRAIL polypeptidecomprising an extracellular domain of human TRAIL of SEQ ID NO: 1,wherein the cell is encapsulated in a matrix or scaffold.
 37. Acomposition of claim 36, further comprising a nucleic acid sequenceencoding HSV-TK.
 38. A composition of claim 36, wherein the solubleTRAIL comprises amino acids 114-281 of SEQ ID NO:
 1. 39. A compositionof claim 36, wherein the soluble TRAIL polypeptide comprises a fusion ofhuman TRAIL polypeptide with an optical imaging reporter polypeptide.40. A composition of claim 36, wherein the matrix comprises a syntheticextracellular matrix.
 41. A composition of claim 36, wherein the matrixis biodegradable.
 42. A composition of claim 40, wherein, wherein thesynthetic extracellular matrix comprises a thiol-modified hyaluronicacid and a thiol-reactive cross-linker molecule.
 43. A composition ofclaim 42, wherein the thiol-reactive cross-linker molecule ispolyethylene glycol diacrylate.